dna methylation Search Results


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  • 99
    Zymo Research ez dna methylation gold kit
    PVX-based expression of C4 reverses TGS of a GFP transgene and suppresses endogenous and exogenous <t>DNA</t> methylation. (A) 16-TGS plants were inoculated with PVX-C4-HA, PVX-C4 R13A -HA or PVX-cLUC-HA, and photographed under UV light at 14 dpi. cLUC represents c-terminal fragment of the firefly luciferase. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as a protein loading control. (C) Real-time <t>RT-PCR</t> showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Values represent means ± SE from three independent experiments. (*p
    Ez Dna Methylation Gold Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 8082 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez dna methylation gold kit/product/Zymo Research
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    99
    Zymo Research ez dna methylation direct kit
    MicroRNA expression analysis of matched fresh and <t>FFPE</t> RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep <t>DNA/RNA</t> FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Ez Dna Methylation Direct Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 2009 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez dna methylation direct kit/product/Zymo Research
    Average 99 stars, based on 2009 article reviews
    Price from $9.99 to $1999.99
    ez dna methylation direct kit - by Bioz Stars, 2019-05
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    99
    Zymo Research ez dna methylation lightning kit
    Direct Bisulfite Conversion of <t>FFPE</t> Tissue Sections without Prior Extraction. Total yield of bisulfite-converted <t>DNA</t> derived from the direct processing of 10 μm FFPE placental tissue sections using three bisulfite conversion kits. Each kit was tested in 9 replicates. Shown are mean values (± standard deviation) of triplicate CFF qPCR measurements ( B ) and results from a single UV measurement ( A ), respectively.
    Ez Dna Methylation Lightning Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 894 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez dna methylation lightning kit/product/Zymo Research
    Average 99 stars, based on 894 article reviews
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    99
    Zymo Research ez dna methylation kit
    Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in <t>DNA</t> from WT and <t>DKO</t> cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software 64 from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. 39 Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.
    Ez Dna Methylation Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 10281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez dna methylation kit/product/Zymo Research
    Average 99 stars, based on 10281 article reviews
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    ez dna methylation kit - by Bioz Stars, 2019-05
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    94
    Illumina Inc truseq dna methylation kit
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared <t>DNA</t> fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The <t>TruSeq</t> DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    Truseq Dna Methylation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/truseq dna methylation kit/product/Illumina Inc
    Average 94 stars, based on 153 article reviews
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    truseq dna methylation kit - by Bioz Stars, 2019-05
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    84
    Cayman Chemical dna methylation elisa kit
    AC with hypomethylated <t>DNA</t> are unable to suppress inflammatory arthritis. DC were left untreated and made apoptotic (AC) or treated with 5-azacytidine and then made apoptotic (AZA AC) and the level of global methylation analyzed by <t>ELISA</t> ( A ). Mice were left untreated (UT) or injected with AC or AZA AC (AZA) at the time of immunization with CFA/mBSA and for a further 2 consecutive days. Arthritis was induced 7 days later and clinical score assessed for a further 7 days ( B ) and knee swelling determined 3 days post knee injections ( C ). 7 days post knee injection, draining lymph nodes were harvested and analyzed for IL-17 production by ELISA ( D ). Graphs shows mean cytokine production ± SEM. Data is pooled from 4 independent experiments with a total of 12 mice per group. Statistical analysis was performed using a t-test ( A , paired; C and D , unpaired) or a one-way analysis of variance (ANOVA) followed by Bonferroni post hoc testing for multiple comparisons ( B ).
    Dna Methylation Elisa Kit, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 84/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna methylation elisa kit/product/Cayman Chemical
    Average 84 stars, based on 8 article reviews
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    dna methylation elisa kit - by Bioz Stars, 2019-05
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    80
    Sequenom dna methylation analysis
    MALDI-TOF-MS <t>DNA</t> methylation analysis. Overview of the MassCLEAVE™ assay. (A) Genomic DNA is bisulphite treated and <t>PCR-tagged</t> to include the T7 promoter sequence. As shown, either top or bottom strand can be used for amplification. Subsequent alkaline phosphatase (SAP) treatment, in vitro transcription using T7 R DNA polymerase and a specific nucleotide mixture plus RNase A cleavage results in specific fragmentation. As exemplified, the top and bottom strands can have markedly different fragmentation patterns. The obtained mixture of fragments can be analyzed by MALDI-TOF-MS. Spectrum peaks representing methylated and unmethylated fragments are used to calculate methylation levels for every fragment. (B) In silico transcript fragmentation. The use of T- or C-cleavage mixtures on either the top or bottom strand of bisulphite-treated DNA can yield quite different fragmentation patterns. Here, the fragmentation of the CpG island of INHBB (chr2:119,998,230-119,998,596) is shown. CpG sites are represented by circles; white circle: methylation call will be obtained in the MassCLEAVE™ assay; crossed gray circle: methylation call will be missed, because fragments mass falls outside of spectral range of analysis; red diagonal line: approximate cleavage site; dotted line: combined methylation call due to overlapping peaks in spectrum. T-cleavage is more informative than C-cleavage when interrogating CpG islands due to RNaseA digestion after every CpG site in the C-cleavage reaction. (C) New schematic representation of DNA methylation data. Due to the specific fragmentation of a transcript, multiple CpG sites can be present within one fragment. Also, fragments with identical masses will show overlapping peaks in the MALDI-TOF-MS spectrum, resulting in a combined DNA methylation call. This new graphical representation of the DNA methylation incorporates all this information. The white circles represent the CpG sites that are analyzed in the MassCLEAVE™ assay. The crossed grey circles represent CpG sites that will be missed in the assay, and the red diagonal lines are the RNase A cleavage sites. The colored circles in the average view indicate the methylation calls given by the assay with the dashed lines linking these calls back to the CpG site(s) within the interrogated sequence. In the detailed view, the relative abundance of unmethylated, partially methylated, and fully methylated molecules is visualized as proportional bars for each fragment (white, gray and black bars, respectively). This view allows an in-depth graphical comparison of samples at the highest resolution possible.
    Dna Methylation Analysis, supplied by Sequenom, used in various techniques. Bioz Stars score: 80/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna methylation analysis/product/Sequenom
    Average 80 stars, based on 19 article reviews
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    dna methylation analysis - by Bioz Stars, 2019-05
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    79
    Illumina Inc array based dna methylation platform data
    <t>DNA</t> methylation downstream of <t>SALL4</t> TSS interferes with RNA polymerase II elongation ( a ) Diagram of SALL4 gene. TSS and exon1–4 are shown. ChIP assays with RNA polymerase II antibody, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0–D10, and SALL4 primers spanning different exons, as indicated. ( b ) RIP assays with RNA polymerase II antibody, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0 and D10, and SALL4 primers spanning exons 1 and 4, as indicated in (a).
    Array Based Dna Methylation Platform Data, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 79/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/array based dna methylation platform data/product/Illumina Inc
    Average 79 stars, based on 19 article reviews
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    Image Search Results


    PVX-based expression of C4 reverses TGS of a GFP transgene and suppresses endogenous and exogenous DNA methylation. (A) 16-TGS plants were inoculated with PVX-C4-HA, PVX-C4 R13A -HA or PVX-cLUC-HA, and photographed under UV light at 14 dpi. cLUC represents c-terminal fragment of the firefly luciferase. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as a protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Values represent means ± SE from three independent experiments. (*p

    Journal: PLoS Pathogens

    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

    doi: 10.1371/journal.ppat.1007282

    Figure Lengend Snippet: PVX-based expression of C4 reverses TGS of a GFP transgene and suppresses endogenous and exogenous DNA methylation. (A) 16-TGS plants were inoculated with PVX-C4-HA, PVX-C4 R13A -HA or PVX-cLUC-HA, and photographed under UV light at 14 dpi. cLUC represents c-terminal fragment of the firefly luciferase. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as a protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Values represent means ± SE from three independent experiments. (*p

    Article Snippet: To improve the efficiency of bisulfite treatment, DNA (1 mg) was digested with a restriction enzyme that cuts outside the region of interest to decrease the size of DNA, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA) in a PCR machine.

    Techniques: Expressing, DNA Methylation Assay, Hemagglutination Assay, Luciferase, Western Blot, Staining, Quantitative RT-PCR

    Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Journal: PLoS Pathogens

    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

    doi: 10.1371/journal.ppat.1007282

    Figure Lengend Snippet: Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Article Snippet: To improve the efficiency of bisulfite treatment, DNA (1 mg) was digested with a restriction enzyme that cuts outside the region of interest to decrease the size of DNA, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA) in a PCR machine.

    Techniques: Infection, Plasmid Preparation, Southern Blot, Labeling, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

    Silencing of NbSAMS2 reverses TGS and PTGS of GFP. (A) 16-TGS plants were co-inoculated with TYLCCNV and βM2 vector containing DNA fragment of NbSAMS2 or LUC. Plants were photographed under UV light at 21 dpi. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as an equal protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Data were obtained from three independent experiments. Values represent means ± SE from three independent experiments. (*p

    Journal: PLoS Pathogens

    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

    doi: 10.1371/journal.ppat.1007282

    Figure Lengend Snippet: Silencing of NbSAMS2 reverses TGS and PTGS of GFP. (A) 16-TGS plants were co-inoculated with TYLCCNV and βM2 vector containing DNA fragment of NbSAMS2 or LUC. Plants were photographed under UV light at 21 dpi. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as an equal protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Data were obtained from three independent experiments. Values represent means ± SE from three independent experiments. (*p

    Article Snippet: To improve the efficiency of bisulfite treatment, DNA (1 mg) was digested with a restriction enzyme that cuts outside the region of interest to decrease the size of DNA, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA) in a PCR machine.

    Techniques: Plasmid Preparation, Western Blot, Staining, Quantitative RT-PCR

    A R13A point mutation in C4 attenuates CLCuMuV infection. (A) Schematic representation of the CLCuMuV C4 and Rep ORF. Sequences in light blue color represents mutation while red color for original nucleotide and relevant amino acids was displayed with green color. (B) Relative viral accumulation of CLCuMuV DNA. Protoplasts from the leaves of N . benthamiana plants were isolated, and then transfected with CLCuMuVΔRep and expression construct of either Rep or RepE65G. Real-time PCR analysis of V1 gene from CLCuMuVΔRep was used to determine viral DNA level. eIF4α was used as an internal control. Values represent means ± SE from three independent experiments. (C) The mutant CLCuMuV carrying C4 R13A (CLCuMuV-C4 R13A ), which contains a R13A mutation in C4 , showed the decreased viral symptom compared to wild type CLCuMuV. Photographs were taken at 21 dpi. (D) Relative viral accumulation of CLCuMuV DNA. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Journal: PLoS Pathogens

    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

    doi: 10.1371/journal.ppat.1007282

    Figure Lengend Snippet: A R13A point mutation in C4 attenuates CLCuMuV infection. (A) Schematic representation of the CLCuMuV C4 and Rep ORF. Sequences in light blue color represents mutation while red color for original nucleotide and relevant amino acids was displayed with green color. (B) Relative viral accumulation of CLCuMuV DNA. Protoplasts from the leaves of N . benthamiana plants were isolated, and then transfected with CLCuMuVΔRep and expression construct of either Rep or RepE65G. Real-time PCR analysis of V1 gene from CLCuMuVΔRep was used to determine viral DNA level. eIF4α was used as an internal control. Values represent means ± SE from three independent experiments. (C) The mutant CLCuMuV carrying C4 R13A (CLCuMuV-C4 R13A ), which contains a R13A mutation in C4 , showed the decreased viral symptom compared to wild type CLCuMuV. Photographs were taken at 21 dpi. (D) Relative viral accumulation of CLCuMuV DNA. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Article Snippet: To improve the efficiency of bisulfite treatment, DNA (1 mg) was digested with a restriction enzyme that cuts outside the region of interest to decrease the size of DNA, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA) in a PCR machine.

    Techniques: Mutagenesis, Infection, Isolation, Transfection, Expressing, Construct, Real-time Polymerase Chain Reaction

    DNA methylation analysis at CTCF binding sites in BCL6 intronic region of primary tumors. (A) Quantitative RT-PCR analysis of BCL6 mRNA abundance in lymphoma and plasma cell myeloma samples. The box-and-whisker plot illustrates the BCL6 expression level of 11 lymphoma and 8 myeloma samples as the percentage of expression level in Raji (y axis is plotted in log scale). The horizontal bar within the box represents the median (50th percentile) of the data points within each sample group, whereas the top and the bottom of the box represent the upper and lower quartile range (75th and 25th percentile), respectively. The whiskers represent the spread of data points within 1.5 interquartile range, and outliers are represented as open circles. (B) MSP analysis of CpGs at CTCF sites 3 and 4 in BCL6 intronic region. Intensities of 32 P signal in the Southern blot of MSP and the corresponding unmethylated-specific PCR (USP) were quantified and represented as MSP/USP intensity ratio. MSP/USP primer locations and the CpGs being analyzed are shown in Fig. S6 B . The box-and-whisker plot compares the MSP/USP intensity ratio of the same lymphoma and myeloma samples analyzed in A.

    Journal: The Journal of Experimental Medicine

    Article Title: DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas

    doi: 10.1084/jem.20100204

    Figure Lengend Snippet: DNA methylation analysis at CTCF binding sites in BCL6 intronic region of primary tumors. (A) Quantitative RT-PCR analysis of BCL6 mRNA abundance in lymphoma and plasma cell myeloma samples. The box-and-whisker plot illustrates the BCL6 expression level of 11 lymphoma and 8 myeloma samples as the percentage of expression level in Raji (y axis is plotted in log scale). The horizontal bar within the box represents the median (50th percentile) of the data points within each sample group, whereas the top and the bottom of the box represent the upper and lower quartile range (75th and 25th percentile), respectively. The whiskers represent the spread of data points within 1.5 interquartile range, and outliers are represented as open circles. (B) MSP analysis of CpGs at CTCF sites 3 and 4 in BCL6 intronic region. Intensities of 32 P signal in the Southern blot of MSP and the corresponding unmethylated-specific PCR (USP) were quantified and represented as MSP/USP intensity ratio. MSP/USP primer locations and the CpGs being analyzed are shown in Fig. S6 B . The box-and-whisker plot compares the MSP/USP intensity ratio of the same lymphoma and myeloma samples analyzed in A.

    Article Snippet: To analyze DNA methylation at the BCL6 locus, genomic DNA from tumor samples were bisulfite treated using the EZ DNA Methylation Gold kit (Zymo Research).

    Techniques: DNA Methylation Assay, Binding Assay, Quantitative RT-PCR, Whisker Assay, Expressing, Southern Blot, Polymerase Chain Reaction

    CTCF binding controls BCL6 expression. (A) The diagram depicts the genomic features of the BCL6 locus, including the locations of hypersensitive sites and the PCR amplicons used in the ChIP analysis. (B) The graph (left) depicts a representative example of CTCF ChIP at the BCL6 locus in Raji and H929. CTCF enrichment at intronic CTCF sites was also analyzed in Raji after 5-Aza-C treatment for 5 d (right). The graph represents mean enrichment levels from two experiments for each treatment condition. Data are presented as the percentage of input for each primer set, determined by quantitative PCR with comparison of immunoprecipitated DNA with a standard curve of DNA purified from input chromatin for each sample. (C) mRNA abundance in H929 cells treated with CTCF shRNA or empty vector was determined by quantitative RT-PCR. Data represent the mean of three independent replicates. Error bars indicate standard deviation.

    Journal: The Journal of Experimental Medicine

    Article Title: DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas

    doi: 10.1084/jem.20100204

    Figure Lengend Snippet: CTCF binding controls BCL6 expression. (A) The diagram depicts the genomic features of the BCL6 locus, including the locations of hypersensitive sites and the PCR amplicons used in the ChIP analysis. (B) The graph (left) depicts a representative example of CTCF ChIP at the BCL6 locus in Raji and H929. CTCF enrichment at intronic CTCF sites was also analyzed in Raji after 5-Aza-C treatment for 5 d (right). The graph represents mean enrichment levels from two experiments for each treatment condition. Data are presented as the percentage of input for each primer set, determined by quantitative PCR with comparison of immunoprecipitated DNA with a standard curve of DNA purified from input chromatin for each sample. (C) mRNA abundance in H929 cells treated with CTCF shRNA or empty vector was determined by quantitative RT-PCR. Data represent the mean of three independent replicates. Error bars indicate standard deviation.

    Article Snippet: To analyze DNA methylation at the BCL6 locus, genomic DNA from tumor samples were bisulfite treated using the EZ DNA Methylation Gold kit (Zymo Research).

    Techniques: Binding Assay, Expressing, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, shRNA, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

    Presence of DNase I hypersensitive sites within CpG islands 32 and 27 in the absence of DNA methylation. (A) The diagram depicts the approximate location of the genomic features of the BCL6 locus along with the restriction fragments and probes used for indirect end labeling. The approximate locations of the observed hypersensitive sites are indicated on the diagram. (B) The 5′ end of the BCL6 locus was analyzed by DNase I digestion using the reference restriction enzyme XbaI. Wedges indicate concentrations of DNase I. The locations of restriction sites and the probe used for the Southern blot are depicted in A. Arrows indicate the major DNase I hypersensitive sites and their locations are summarized in A. Marker positions were measured from ethidium-stained gel before transfer. Data shown is representative of results from at least three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas

    doi: 10.1084/jem.20100204

    Figure Lengend Snippet: Presence of DNase I hypersensitive sites within CpG islands 32 and 27 in the absence of DNA methylation. (A) The diagram depicts the approximate location of the genomic features of the BCL6 locus along with the restriction fragments and probes used for indirect end labeling. The approximate locations of the observed hypersensitive sites are indicated on the diagram. (B) The 5′ end of the BCL6 locus was analyzed by DNase I digestion using the reference restriction enzyme XbaI. Wedges indicate concentrations of DNase I. The locations of restriction sites and the probe used for the Southern blot are depicted in A. Arrows indicate the major DNase I hypersensitive sites and their locations are summarized in A. Marker positions were measured from ethidium-stained gel before transfer. Data shown is representative of results from at least three independent experiments.

    Article Snippet: To analyze DNA methylation at the BCL6 locus, genomic DNA from tumor samples were bisulfite treated using the EZ DNA Methylation Gold kit (Zymo Research).

    Techniques: DNA Methylation Assay, End Labeling, Southern Blot, Marker, Staining

    Cell type–specific DNA methylation status at BCL6 intronic region. (A) The diagram depicts the genomic organization of the human BCL6 locus. Indicated are the two transcription start sites (arrows), the four CpG islands (17, 32, 27, and 38), and the exons (black rectangles). Below the cartoon, the results of genomic bisulfite sequencing are presented. Each line of circles indicates an individual clone sequenced in the analysis after bisulfite treatment and PCR. Open circles indicate CpG sites at which no DNA methylation is detected. Blackened circles indicate CpG sites which are methylated. Data shown is representative of the results of three independent biological replicates. (B) MIRA-chip analysis of Raji (top) and H929 (bottom) across the 5′ end of the BCL6 locus. Each vertical line represents the mean normalized log 2 ratio of enriched/input probe signal from two replicate samples, corresponding to the genomic location on chromosome 3 at the BCL6 locus (UCSC genome browser HG18) as listed on the x axis. Blue and yellow colors represent methylated and unmethylated regions, respectively.

    Journal: The Journal of Experimental Medicine

    Article Title: DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas

    doi: 10.1084/jem.20100204

    Figure Lengend Snippet: Cell type–specific DNA methylation status at BCL6 intronic region. (A) The diagram depicts the genomic organization of the human BCL6 locus. Indicated are the two transcription start sites (arrows), the four CpG islands (17, 32, 27, and 38), and the exons (black rectangles). Below the cartoon, the results of genomic bisulfite sequencing are presented. Each line of circles indicates an individual clone sequenced in the analysis after bisulfite treatment and PCR. Open circles indicate CpG sites at which no DNA methylation is detected. Blackened circles indicate CpG sites which are methylated. Data shown is representative of the results of three independent biological replicates. (B) MIRA-chip analysis of Raji (top) and H929 (bottom) across the 5′ end of the BCL6 locus. Each vertical line represents the mean normalized log 2 ratio of enriched/input probe signal from two replicate samples, corresponding to the genomic location on chromosome 3 at the BCL6 locus (UCSC genome browser HG18) as listed on the x axis. Blue and yellow colors represent methylated and unmethylated regions, respectively.

    Article Snippet: To analyze DNA methylation at the BCL6 locus, genomic DNA from tumor samples were bisulfite treated using the EZ DNA Methylation Gold kit (Zymo Research).

    Techniques: DNA Methylation Assay, Methylation Sequencing, Polymerase Chain Reaction, Methylation, Chromatin Immunoprecipitation

    Positive regulation of BCL6 transcription by DNA methylation. (A) The Raji, HsSultan, and EB1 Burkitt lymphoma cell lines were treated with 5-Aza-C for 24 h, and BCL6 and actin mRNA abundance was determined by quantitative RT-PCR. The bar graph depicts the BCL6 to actin ratio from three independent replicates. Error bars indicate standard deviation. The value from untreated cells for each replicate was arbitrarily set to 1. (B) Raji cells were treated with 5-Aza-C for the indicated times. BCL6 , IRF4 , and PRDM1 mRNA abundance was determined by quantitative RT-PCR. The bar graphs depict the ratio of each transcript to GAPDH mRNA, with the value from untreated cells (day 0) arbitrarily set to 1. Data represent the mean of three independent replicates. Error bars indicate standard deviation. (C) The diagram depicts the basic features of the BCL6 locus 5′ end along with the approximate locations of primer sets used to analyze the chromatin immunoprecipitated with the indicated antibodies in each panel. Each ChIP primer was analyzed by quantitative PCR with the graph depicting the percentage of input chromatin recovered in the immunoprecipitation for each primer set. The data represent the mean of two independent biological replicates.

    Journal: The Journal of Experimental Medicine

    Article Title: DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas

    doi: 10.1084/jem.20100204

    Figure Lengend Snippet: Positive regulation of BCL6 transcription by DNA methylation. (A) The Raji, HsSultan, and EB1 Burkitt lymphoma cell lines were treated with 5-Aza-C for 24 h, and BCL6 and actin mRNA abundance was determined by quantitative RT-PCR. The bar graph depicts the BCL6 to actin ratio from three independent replicates. Error bars indicate standard deviation. The value from untreated cells for each replicate was arbitrarily set to 1. (B) Raji cells were treated with 5-Aza-C for the indicated times. BCL6 , IRF4 , and PRDM1 mRNA abundance was determined by quantitative RT-PCR. The bar graphs depict the ratio of each transcript to GAPDH mRNA, with the value from untreated cells (day 0) arbitrarily set to 1. Data represent the mean of three independent replicates. Error bars indicate standard deviation. (C) The diagram depicts the basic features of the BCL6 locus 5′ end along with the approximate locations of primer sets used to analyze the chromatin immunoprecipitated with the indicated antibodies in each panel. Each ChIP primer was analyzed by quantitative PCR with the graph depicting the percentage of input chromatin recovered in the immunoprecipitation for each primer set. The data represent the mean of two independent biological replicates.

    Article Snippet: To analyze DNA methylation at the BCL6 locus, genomic DNA from tumor samples were bisulfite treated using the EZ DNA Methylation Gold kit (Zymo Research).

    Techniques: DNA Methylation Assay, Quantitative RT-PCR, Standard Deviation, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    DNA methyltransferase 1 (DNMT 1) inhibitor demethylated the Klotho gene and increased Klotho expression in vivo . ( a ) Methylation-specific PCR (MSP) with mouse Klotho set 1 primers showed that simultaneous treatment with 5-aza-2′-deoxycytidine (5Aza-2dc) in indoxyl sulfate (IS)- and p -cresyl sulfate (PCS)-injected mice significantly demethylated the Klotho gene. ( b ) Western blotting showed that 5Aza-2dc significantly increased the Klotho expression in IS- and PCS-injected mice.

    Journal: Kidney International

    Article Title: Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation

    doi: 10.1038/ki.2011.445

    Figure Lengend Snippet: DNA methyltransferase 1 (DNMT 1) inhibitor demethylated the Klotho gene and increased Klotho expression in vivo . ( a ) Methylation-specific PCR (MSP) with mouse Klotho set 1 primers showed that simultaneous treatment with 5-aza-2′-deoxycytidine (5Aza-2dc) in indoxyl sulfate (IS)- and p -cresyl sulfate (PCS)-injected mice significantly demethylated the Klotho gene. ( b ) Western blotting showed that 5Aza-2dc significantly increased the Klotho expression in IS- and PCS-injected mice.

    Article Snippet: The genomic DNA was extracted (Quick-gDNA MiniPrep, Zymo Research, Irvine, CA) and modified by bisulfate treatment (EZ DNA Methylation-Gold Kit, Zymo Research) for MSP analyses with two sets of gene promoter–specific primer pairs that recognize the methylated and unmethylated CpG sites.

    Techniques: Expressing, In Vivo, Methylation, Polymerase Chain Reaction, Injection, Mouse Assay, Western Blot

    Indoxyl sulfate (IS) and p -cresyl sulfate (PCS) increased DNA methylation of the Klotho gene in vitro . Chromosome DNA samples from cultured HK2 cells treated with IS or PCS for 72 h were analyzed. Methylation-specific PCR (MSP) with human Klotho primer sets ( a ) 1 and ( b ) 2 revealed that the methylation indexes of HK2 cells treated with IS or PCS were significantly increased. * P

    Journal: Kidney International

    Article Title: Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation

    doi: 10.1038/ki.2011.445

    Figure Lengend Snippet: Indoxyl sulfate (IS) and p -cresyl sulfate (PCS) increased DNA methylation of the Klotho gene in vitro . Chromosome DNA samples from cultured HK2 cells treated with IS or PCS for 72 h were analyzed. Methylation-specific PCR (MSP) with human Klotho primer sets ( a ) 1 and ( b ) 2 revealed that the methylation indexes of HK2 cells treated with IS or PCS were significantly increased. * P

    Article Snippet: The genomic DNA was extracted (Quick-gDNA MiniPrep, Zymo Research, Irvine, CA) and modified by bisulfate treatment (EZ DNA Methylation-Gold Kit, Zymo Research) for MSP analyses with two sets of gene promoter–specific primer pairs that recognize the methylated and unmethylated CpG sites.

    Techniques: DNA Methylation Assay, In Vitro, Cell Culture, Methylation, Polymerase Chain Reaction

    Indoxyl sulfate (IS)- and p -cresyl sulfate (PCS)-injected mice had increased DNA methyltransferase 1 (DNMT 1) expression and DNA hypermethylation of the Klotho gene. ( a ) The results of western blot analysis and immunostaining showed that IS and PCS significantly increased the DNMT 1 expression. ( b ) Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of Klotho genes than control mice. * P

    Journal: Kidney International

    Article Title: Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation

    doi: 10.1038/ki.2011.445

    Figure Lengend Snippet: Indoxyl sulfate (IS)- and p -cresyl sulfate (PCS)-injected mice had increased DNA methyltransferase 1 (DNMT 1) expression and DNA hypermethylation of the Klotho gene. ( a ) The results of western blot analysis and immunostaining showed that IS and PCS significantly increased the DNMT 1 expression. ( b ) Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of Klotho genes than control mice. * P

    Article Snippet: The genomic DNA was extracted (Quick-gDNA MiniPrep, Zymo Research, Irvine, CA) and modified by bisulfate treatment (EZ DNA Methylation-Gold Kit, Zymo Research) for MSP analyses with two sets of gene promoter–specific primer pairs that recognize the methylated and unmethylated CpG sites.

    Techniques: Injection, Mouse Assay, Expressing, Western Blot, Immunostaining, Methylation, Polymerase Chain Reaction

    DNA methyltransferase 1 (DNMT 1) inhibitor demethylated the Klotho gene. Methylation-specific PCR (MSP) with human Klotho set 1 primers showed that 5-Aza-2′-deoxycytidine (5Aza-2dc) inhibited Klotho gene hypermethylation in cultured HK2 cells treated with 50 mg/l indoxyl sulfate (IS) or p -cresyl sulfate (PCS). * P

    Journal: Kidney International

    Article Title: Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation

    doi: 10.1038/ki.2011.445

    Figure Lengend Snippet: DNA methyltransferase 1 (DNMT 1) inhibitor demethylated the Klotho gene. Methylation-specific PCR (MSP) with human Klotho set 1 primers showed that 5-Aza-2′-deoxycytidine (5Aza-2dc) inhibited Klotho gene hypermethylation in cultured HK2 cells treated with 50 mg/l indoxyl sulfate (IS) or p -cresyl sulfate (PCS). * P

    Article Snippet: The genomic DNA was extracted (Quick-gDNA MiniPrep, Zymo Research, Irvine, CA) and modified by bisulfate treatment (EZ DNA Methylation-Gold Kit, Zymo Research) for MSP analyses with two sets of gene promoter–specific primer pairs that recognize the methylated and unmethylated CpG sites.

    Techniques: Methylation, Polymerase Chain Reaction, Cell Culture

    MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Article Snippet: Sodium bisulfite treatment was performed with 100–200 ng of fresh and FFPE-DNA using the EZ DNA methylation direct kit (Zymo Research, CA, USA), following manufacturers' protocol.

    Techniques: Expressing, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Article Snippet: Sodium bisulfite treatment was performed with 100–200 ng of fresh and FFPE-DNA using the EZ DNA methylation direct kit (Zymo Research, CA, USA), following manufacturers' protocol.

    Techniques: Formalin-fixed Paraffin-Embedded, Cell Culture, RNA Extraction, Isolation, Agarose Gel Electrophoresis

    DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.

    Article Snippet: Sodium bisulfite treatment was performed with 100–200 ng of fresh and FFPE-DNA using the EZ DNA methylation direct kit (Zymo Research, CA, USA), following manufacturers' protocol.

    Techniques: Formalin-fixed Paraffin-Embedded, RNA Extraction, Isolation

    Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.

    Article Snippet: Sodium bisulfite treatment was performed with 100–200 ng of fresh and FFPE-DNA using the EZ DNA methylation direct kit (Zymo Research, CA, USA), following manufacturers' protocol.

    Techniques: Methylation, Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Electrophoresis, Polymerase Chain Reaction, Isolation, DNA Methylation Assay

    Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.

    Article Snippet: Sodium bisulfite treatment was performed with 100–200 ng of fresh and FFPE-DNA using the EZ DNA methylation direct kit (Zymo Research, CA, USA), following manufacturers' protocol.

    Techniques: Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Electrophoresis

    Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Article Snippet: Sodium bisulfite treatment was performed with 100–200 ng of fresh and FFPE-DNA using the EZ DNA methylation direct kit (Zymo Research, CA, USA), following manufacturers' protocol.

    Techniques: RNA Expression, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    DNA methylation of the putative aromatase promoter at the individual CpG site in the gonads. (A) DNA methylation status in clones from the gonads at embryonic stage 16, 19, and 21 at MPT and FPT. Each row represents a single clone (1–20∼22 clones/stage/temperature) and a column represents the position of CpG (I – VII). Open circles: unmethylated CpG sites. Closed circles: methylated CpG sites. (B) Percent DNA methylation at the individual CpG site (I – VII) of the putative aromatase promoter at stage 16, 19, and 21 in the slider gonad. Asterisk (*) is statistically significant (p

    Journal: PLoS ONE

    Article Title: Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles

    doi: 10.1371/journal.pone.0063599

    Figure Lengend Snippet: DNA methylation of the putative aromatase promoter at the individual CpG site in the gonads. (A) DNA methylation status in clones from the gonads at embryonic stage 16, 19, and 21 at MPT and FPT. Each row represents a single clone (1–20∼22 clones/stage/temperature) and a column represents the position of CpG (I – VII). Open circles: unmethylated CpG sites. Closed circles: methylated CpG sites. (B) Percent DNA methylation at the individual CpG site (I – VII) of the putative aromatase promoter at stage 16, 19, and 21 in the slider gonad. Asterisk (*) is statistically significant (p

    Article Snippet: DNA methylation status in the region of gonad-specific TSS (between −571 and +23 relative to the position of TSS counted as +1) of the slider aromatase gene was examined using EZ DNA Methylation-Direct kit (Zymo research, Irvine, CA).

    Techniques: DNA Methylation Assay, Clone Assay, Methylation

    Sequence homology analysis of the 5′-flanking region of the aromatase in the red-eared slider turtle. The newly identified 4113 bp sequence of the aromatase in the red-eared slider turtle (GenBank accession no. KC554066), consisting of 4066 bp upstream and 47 bp downstream of the translation start codon (ATG), was pair-aligned with the aromatase sequences (upstream bp length from ATG/total bp sequence compared) of the American alligator (3950/4000), chicken (2614/3229), quail (2442/4351), and zebra finch (1356/1401) using the percentage identity plot (PIP) Maker [24] . GenBank accession numbers are as follows: alligator (AY029233), chicken (D50335), quail (D50336), and zebra finch (AH008871). The additional 5′ flanking region of alligator aromatase was extracted from the assembled genome [68] . The horizontal axis represents the base-pair position of the aromatase sequence of the red-eared slider turtle, and the vertical axis represents the percent nucleotide similarity of each aligned segment (i.e., stretch of DNA without any gaps) with the corresponding species.

    Journal: PLoS ONE

    Article Title: Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles

    doi: 10.1371/journal.pone.0063599

    Figure Lengend Snippet: Sequence homology analysis of the 5′-flanking region of the aromatase in the red-eared slider turtle. The newly identified 4113 bp sequence of the aromatase in the red-eared slider turtle (GenBank accession no. KC554066), consisting of 4066 bp upstream and 47 bp downstream of the translation start codon (ATG), was pair-aligned with the aromatase sequences (upstream bp length from ATG/total bp sequence compared) of the American alligator (3950/4000), chicken (2614/3229), quail (2442/4351), and zebra finch (1356/1401) using the percentage identity plot (PIP) Maker [24] . GenBank accession numbers are as follows: alligator (AY029233), chicken (D50335), quail (D50336), and zebra finch (AH008871). The additional 5′ flanking region of alligator aromatase was extracted from the assembled genome [68] . The horizontal axis represents the base-pair position of the aromatase sequence of the red-eared slider turtle, and the vertical axis represents the percent nucleotide similarity of each aligned segment (i.e., stretch of DNA without any gaps) with the corresponding species.

    Article Snippet: DNA methylation status in the region of gonad-specific TSS (between −571 and +23 relative to the position of TSS counted as +1) of the slider aromatase gene was examined using EZ DNA Methylation-Direct kit (Zymo research, Irvine, CA).

    Techniques: Sequencing

    DNA methylation of the putative aromatase promoter at the individual CpG site in temperature-shifted gonads. At embryonic stage 16, eggs are shifted to the opposite temperature, MPT to FPT (A, C) and FPT to MPT (B, D). (A, B) DNA methylation status in clones from the gonads at embryonic stage 19 and 21 after temperature-shift. Each row represents a single clone (1–21∼22 clones/stage/temperature) and a column represents the position of CpG (I – VII). Open circles: unmethylated CpG sites. Closed circles: methylated CpG sites. (C, D) Percent DNA methylation at the individual CpG site (I – VII) of the putative aromatase promoter at stage 19 and 21 in the slider gonad. Asterisk (*) is statistically significant (p

    Journal: PLoS ONE

    Article Title: Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles

    doi: 10.1371/journal.pone.0063599

    Figure Lengend Snippet: DNA methylation of the putative aromatase promoter at the individual CpG site in temperature-shifted gonads. At embryonic stage 16, eggs are shifted to the opposite temperature, MPT to FPT (A, C) and FPT to MPT (B, D). (A, B) DNA methylation status in clones from the gonads at embryonic stage 19 and 21 after temperature-shift. Each row represents a single clone (1–21∼22 clones/stage/temperature) and a column represents the position of CpG (I – VII). Open circles: unmethylated CpG sites. Closed circles: methylated CpG sites. (C, D) Percent DNA methylation at the individual CpG site (I – VII) of the putative aromatase promoter at stage 19 and 21 in the slider gonad. Asterisk (*) is statistically significant (p

    Article Snippet: DNA methylation status in the region of gonad-specific TSS (between −571 and +23 relative to the position of TSS counted as +1) of the slider aromatase gene was examined using EZ DNA Methylation-Direct kit (Zymo research, Irvine, CA).

    Techniques: DNA Methylation Assay, Clone Assay, Methylation

    Putative aromatase promoter (−583 to +170) sequence in the gonads. The core conserved sequences (four nucleotides) for transcription factor binding sites (TFBSs) and CpG sites (I – VII) are indicated in colored and gray boxes, respectively. The gonad-specific transcription start site (TSS, +1) is indicated with an arrow. The translation initiation codon, ATG, is indicated in bold. The sequences of the Arom Set5 primer pairs used for DNA methylation analysis are underlined. SF1: vertebrate steroidogenic factor 1. ERE: estrogen response elements. FOX: fork head domain factors. DM: DM domain-containing transcription factor. SOX: Sox/SRY sex/testis-determining and related HMG box factors. HEAT: heat shock factors. TATA: TATA box.

    Journal: PLoS ONE

    Article Title: Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles

    doi: 10.1371/journal.pone.0063599

    Figure Lengend Snippet: Putative aromatase promoter (−583 to +170) sequence in the gonads. The core conserved sequences (four nucleotides) for transcription factor binding sites (TFBSs) and CpG sites (I – VII) are indicated in colored and gray boxes, respectively. The gonad-specific transcription start site (TSS, +1) is indicated with an arrow. The translation initiation codon, ATG, is indicated in bold. The sequences of the Arom Set5 primer pairs used for DNA methylation analysis are underlined. SF1: vertebrate steroidogenic factor 1. ERE: estrogen response elements. FOX: fork head domain factors. DM: DM domain-containing transcription factor. SOX: Sox/SRY sex/testis-determining and related HMG box factors. HEAT: heat shock factors. TATA: TATA box.

    Article Snippet: DNA methylation status in the region of gonad-specific TSS (between −571 and +23 relative to the position of TSS counted as +1) of the slider aromatase gene was examined using EZ DNA Methylation-Direct kit (Zymo research, Irvine, CA).

    Techniques: Sequencing, Binding Assay, DNA Methylation Assay

    Total DNA methylation level of the putative aromatase promoter in the gonads. Total percent DNA methylation during embryonic development at MPT and FPT (A), MPT shifted to FPT (C), and FPT shifted to MPT (D) were examined. (B) The scheme of temperature-shift experiment. Each bar represents twenty to twenty-two clones/stage/temperature from the data of a single pool of 10 gonads. Asterisk (*) is statistically significant (p

    Journal: PLoS ONE

    Article Title: Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles

    doi: 10.1371/journal.pone.0063599

    Figure Lengend Snippet: Total DNA methylation level of the putative aromatase promoter in the gonads. Total percent DNA methylation during embryonic development at MPT and FPT (A), MPT shifted to FPT (C), and FPT shifted to MPT (D) were examined. (B) The scheme of temperature-shift experiment. Each bar represents twenty to twenty-two clones/stage/temperature from the data of a single pool of 10 gonads. Asterisk (*) is statistically significant (p

    Article Snippet: DNA methylation status in the region of gonad-specific TSS (between −571 and +23 relative to the position of TSS counted as +1) of the slider aromatase gene was examined using EZ DNA Methylation-Direct kit (Zymo research, Irvine, CA).

    Techniques: DNA Methylation Assay, Clone Assay

    DNA methylation status of Snrpn , Lep , and Ppar-α in oocytes from CD and HFD dams as analyzed by bisulfite sequencing. Oocytes from 10 mice were used per analysis. ( A ) DNA methylation level of Snrpn ( A ; red arrowheads indicate the recognition sites of BstUI ), and Lep ( B ; analyzed region located at chr6: 26009934..26010283). Numbers indicate the percentage of methylation; blank loci indicate lost CpG. ( C ) Distribution of some CpG sites in the Ppar-α promoter in the analyzed region; CpG sites are numbered 1–14. ( D ) Percentage of DNA methylation at the 14 CpG sites in the Ppar-α promoter ( C ).

    Journal: Environmental Health Perspectives

    Article Title: DNA Methylation in Oocytes and Liver of Female Mice and Their Offspring: Effects of High-Fat-Diet-Induced Obesity

    doi: 10.1289/ehp.1307047

    Figure Lengend Snippet: DNA methylation status of Snrpn , Lep , and Ppar-α in oocytes from CD and HFD dams as analyzed by bisulfite sequencing. Oocytes from 10 mice were used per analysis. ( A ) DNA methylation level of Snrpn ( A ; red arrowheads indicate the recognition sites of BstUI ), and Lep ( B ; analyzed region located at chr6: 26009934..26010283). Numbers indicate the percentage of methylation; blank loci indicate lost CpG. ( C ) Distribution of some CpG sites in the Ppar-α promoter in the analyzed region; CpG sites are numbered 1–14. ( D ) Percentage of DNA methylation at the 14 CpG sites in the Ppar-α promoter ( C ).

    Article Snippet: Liver DNA from HFD offspring (OHFD) and CD offspring (OCD) was modified using the EZ DNA Methylation-Direct™ Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Methylation Sequencing, Mouse Assay, Methylation

    DNA methylation patterns in DMRs of paternally imprinted gene H19 ( A ) and maternally imprinted genes Igf2r ( B ), Peg1 ( C ), Peg3 ( D ), and Snrpn ( E ) in oocytes from CD and HFD dams as determined by COBRA. Oocytes from 10 mice were used per analysis. Spermatozoa ( S ) were used as a control. Restriction enzymes used are shown on the right. Red arrowheads indicate undigested bands: For H19 ( A ), the spermatozoa sample was digested and oocyte samples were undigested; for Igf2r ( B ), Peg1 ( C ), Peg3 ( D ), and Snrpn ( E ), the spermatozoa sample was undigested, but some oocyte samples were digested.

    Journal: Environmental Health Perspectives

    Article Title: DNA Methylation in Oocytes and Liver of Female Mice and Their Offspring: Effects of High-Fat-Diet-Induced Obesity

    doi: 10.1289/ehp.1307047

    Figure Lengend Snippet: DNA methylation patterns in DMRs of paternally imprinted gene H19 ( A ) and maternally imprinted genes Igf2r ( B ), Peg1 ( C ), Peg3 ( D ), and Snrpn ( E ) in oocytes from CD and HFD dams as determined by COBRA. Oocytes from 10 mice were used per analysis. Spermatozoa ( S ) were used as a control. Restriction enzymes used are shown on the right. Red arrowheads indicate undigested bands: For H19 ( A ), the spermatozoa sample was digested and oocyte samples were undigested; for Igf2r ( B ), Peg1 ( C ), Peg3 ( D ), and Snrpn ( E ), the spermatozoa sample was undigested, but some oocyte samples were digested.

    Article Snippet: Liver DNA from HFD offspring (OHFD) and CD offspring (OCD) was modified using the EZ DNA Methylation-Direct™ Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, Mouse Assay

    Lep and Ppar-α methylation status and gene expression in the liver of female and male OCD and OHFD mice at 7–8 weeks of age ( n = 10 mice from five litters per sex per group). DNA methylation was analyzed by bisulfite sequencing, and gene expression was evaluated by qRT‑PCR. ( A,B ) DNA methylation of Lep in liver of female ( A ) and male ( B ) offspring. Numbers indicate the percentage of methylation; blank loci indicate lost CpG. ( C,D ) DNA methylation at CpG sites of Ppar-α in liver of female ( C ) and male ( D ) offspring. CpG sites are numbered 1–14. ( E,F ) Expression of Lep and Ppar-α in liver of female ( E ) and male ( F ) offspring. * p

    Journal: Environmental Health Perspectives

    Article Title: DNA Methylation in Oocytes and Liver of Female Mice and Their Offspring: Effects of High-Fat-Diet-Induced Obesity

    doi: 10.1289/ehp.1307047

    Figure Lengend Snippet: Lep and Ppar-α methylation status and gene expression in the liver of female and male OCD and OHFD mice at 7–8 weeks of age ( n = 10 mice from five litters per sex per group). DNA methylation was analyzed by bisulfite sequencing, and gene expression was evaluated by qRT‑PCR. ( A,B ) DNA methylation of Lep in liver of female ( A ) and male ( B ) offspring. Numbers indicate the percentage of methylation; blank loci indicate lost CpG. ( C,D ) DNA methylation at CpG sites of Ppar-α in liver of female ( C ) and male ( D ) offspring. CpG sites are numbered 1–14. ( E,F ) Expression of Lep and Ppar-α in liver of female ( E ) and male ( F ) offspring. * p

    Article Snippet: Liver DNA from HFD offspring (OHFD) and CD offspring (OCD) was modified using the EZ DNA Methylation-Direct™ Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.

    Techniques: Methylation, Expressing, Mouse Assay, DNA Methylation Assay, Methylation Sequencing

    DNA methylation patterns in DMRs of paternally imprinted gene H19 ( A ) and maternally imprinted genes Igf2r ( B ), Peg3 ( C ), and Snrpn ( D ) was in oocytes of OHFD and OCD mice as determined by COBRA. Oocytes from 10 mice were used for each analysis. Spermatozoa ( S ) were used as a control. Restriction enzymes used are shown on the right. Red arrowheads indicate undigested bands.

    Journal: Environmental Health Perspectives

    Article Title: DNA Methylation in Oocytes and Liver of Female Mice and Their Offspring: Effects of High-Fat-Diet-Induced Obesity

    doi: 10.1289/ehp.1307047

    Figure Lengend Snippet: DNA methylation patterns in DMRs of paternally imprinted gene H19 ( A ) and maternally imprinted genes Igf2r ( B ), Peg3 ( C ), and Snrpn ( D ) was in oocytes of OHFD and OCD mice as determined by COBRA. Oocytes from 10 mice were used for each analysis. Spermatozoa ( S ) were used as a control. Restriction enzymes used are shown on the right. Red arrowheads indicate undigested bands.

    Article Snippet: Liver DNA from HFD offspring (OHFD) and CD offspring (OCD) was modified using the EZ DNA Methylation-Direct™ Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Mouse Assay, Combined Bisulfite Restriction Analysis Assay

    DNA methylation status of Snrpn , Lep , IAP (intracisternal A particle), and Ppar-α in oocytes of offspring ( A–D ) and of IAP in oocytes of CD and HFD dams ( E ), as analyzed by bisulfite sequencing. Oocytes from 10 mice were used per analysis. ( A–B ) Methylation status of Snrpn ( A ) and Lep ( B ) in offspring oocytes. ( C ) DNA methylation at CpG sites of Ppar-α in offspring oocytes; CpG sites are numbered 1–14. ( D ) Methylation status of IAP in offspring oocytes. ( E ) Methylation patterns of IAP in oocytes from CD and HFD dams. In ( A,B,D,E ), numbers indicate the percentage of methylation, and blank loci indicate lost CpG. * p

    Journal: Environmental Health Perspectives

    Article Title: DNA Methylation in Oocytes and Liver of Female Mice and Their Offspring: Effects of High-Fat-Diet-Induced Obesity

    doi: 10.1289/ehp.1307047

    Figure Lengend Snippet: DNA methylation status of Snrpn , Lep , IAP (intracisternal A particle), and Ppar-α in oocytes of offspring ( A–D ) and of IAP in oocytes of CD and HFD dams ( E ), as analyzed by bisulfite sequencing. Oocytes from 10 mice were used per analysis. ( A–B ) Methylation status of Snrpn ( A ) and Lep ( B ) in offspring oocytes. ( C ) DNA methylation at CpG sites of Ppar-α in offspring oocytes; CpG sites are numbered 1–14. ( D ) Methylation status of IAP in offspring oocytes. ( E ) Methylation patterns of IAP in oocytes from CD and HFD dams. In ( A,B,D,E ), numbers indicate the percentage of methylation, and blank loci indicate lost CpG. * p

    Article Snippet: Liver DNA from HFD offspring (OHFD) and CD offspring (OCD) was modified using the EZ DNA Methylation-Direct™ Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Methylation Sequencing, Mouse Assay, Methylation

    Direct Bisulfite Conversion of FFPE Tissue Sections without Prior Extraction. Total yield of bisulfite-converted DNA derived from the direct processing of 10 μm FFPE placental tissue sections using three bisulfite conversion kits. Each kit was tested in 9 replicates. Shown are mean values (± standard deviation) of triplicate CFF qPCR measurements ( B ) and results from a single UV measurement ( A ), respectively.

    Journal: PLoS ONE

    Article Title: Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

    doi: 10.1371/journal.pone.0093933

    Figure Lengend Snippet: Direct Bisulfite Conversion of FFPE Tissue Sections without Prior Extraction. Total yield of bisulfite-converted DNA derived from the direct processing of 10 μm FFPE placental tissue sections using three bisulfite conversion kits. Each kit was tested in 9 replicates. Shown are mean values (± standard deviation) of triplicate CFF qPCR measurements ( B ) and results from a single UV measurement ( A ), respectively.

    Article Snippet: Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits).

    Techniques: Formalin-fixed Paraffin-Embedded, Derivative Assay, Standard Deviation, Real-time Polymerase Chain Reaction

    PCR Inhibition and Storage Stability. A: Inhibitory effect of eluate derived from different bisulfite conversion kits. Water was applied to nine different bisulfite conversion kits and processed like sample DNAs (process negative control sample). Different volumes (0–10μl) of this eluate were spiked into the ACTB / SEPT9 / SHOX2 q PCR (20 μl total PCR volume). Each PCR contained 10 ng bisulfite-converted methylated template DNA. B, C: Storage stability. Bisulfite-converted DNA was prepared from HMW ( B ) and FFPE tissue ( C ) input DNA using nine different bisulfite conversion kits. The bisulfite-converted DNA was stored for one month at −80°C, −20°C, 4°C, and 37°C, respectively. Total amount of intact (PCR-amplifiable) DNA was determined using the CFF qPCR assay. Each kit was tested in nine replicates. Each PCR was run in duplicate. Shown are mean values (± standard deviation).

    Journal: PLoS ONE

    Article Title: Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

    doi: 10.1371/journal.pone.0093933

    Figure Lengend Snippet: PCR Inhibition and Storage Stability. A: Inhibitory effect of eluate derived from different bisulfite conversion kits. Water was applied to nine different bisulfite conversion kits and processed like sample DNAs (process negative control sample). Different volumes (0–10μl) of this eluate were spiked into the ACTB / SEPT9 / SHOX2 q PCR (20 μl total PCR volume). Each PCR contained 10 ng bisulfite-converted methylated template DNA. B, C: Storage stability. Bisulfite-converted DNA was prepared from HMW ( B ) and FFPE tissue ( C ) input DNA using nine different bisulfite conversion kits. The bisulfite-converted DNA was stored for one month at −80°C, −20°C, 4°C, and 37°C, respectively. Total amount of intact (PCR-amplifiable) DNA was determined using the CFF qPCR assay. Each kit was tested in nine replicates. Each PCR was run in duplicate. Shown are mean values (± standard deviation).

    Article Snippet: Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits).

    Techniques: Polymerase Chain Reaction, Inhibition, Derivative Assay, Negative Control, Methylation, Formalin-fixed Paraffin-Embedded, Real-time Polymerase Chain Reaction, Standard Deviation

    Kit Performance Comparison Using a Triplex qPCR ( ACTB / SHOX2 / SEPT9 ) Assay. HMW ( upper panel ) and FFPE tissue ( lower panel ) DNA was bisulfite-converted using nine different commercially available kits. Each bisulfite-converted DNA was assayed using the ACTB / SHOX2 / SEPT9 triplex qPCR asay. Shown are mean (± standard deviation) CT values of triplicate measurements. Left panel: ACTB CTs; middle panel: SEPT9 CTs, right panel: SHOX2 CTs.

    Journal: PLoS ONE

    Article Title: Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

    doi: 10.1371/journal.pone.0093933

    Figure Lengend Snippet: Kit Performance Comparison Using a Triplex qPCR ( ACTB / SHOX2 / SEPT9 ) Assay. HMW ( upper panel ) and FFPE tissue ( lower panel ) DNA was bisulfite-converted using nine different commercially available kits. Each bisulfite-converted DNA was assayed using the ACTB / SHOX2 / SEPT9 triplex qPCR asay. Shown are mean (± standard deviation) CT values of triplicate measurements. Left panel: ACTB CTs; middle panel: SEPT9 CTs, right panel: SHOX2 CTs.

    Article Snippet: Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits).

    Techniques: Real-time Polymerase Chain Reaction, Formalin-fixed Paraffin-Embedded, Standard Deviation

    DNA Integrity after Bisulfite Conversion. Agarose gel electrophoresis of genomic and bisulfite-converted DNA (2 μg each). The bisulfite conversion was carried out using nine different kits. Shown is bisulfite-converted DNA from HMW (left) and FFPE tissue (right) input DNA. Each bisulfite-converted DNA represents a DNA pool from nine independent bisulfite reactions per kit.

    Journal: PLoS ONE

    Article Title: Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

    doi: 10.1371/journal.pone.0093933

    Figure Lengend Snippet: DNA Integrity after Bisulfite Conversion. Agarose gel electrophoresis of genomic and bisulfite-converted DNA (2 μg each). The bisulfite conversion was carried out using nine different kits. Shown is bisulfite-converted DNA from HMW (left) and FFPE tissue (right) input DNA. Each bisulfite-converted DNA represents a DNA pool from nine independent bisulfite reactions per kit.

    Article Snippet: Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits).

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Formalin-fixed Paraffin-Embedded

    DNA Yield and Purity after Bisulfite Conversion Applying Nine Different Kits. HMW ( upper panel ) and FFPE tissue ( lower panel ) DNA was bisulfite-converted using nine different commercially available kits. Each bisulfite conversion was conducted in nine replicates for each kit. Left panel: UV spectra of bisulfite-converted and purified DNA ( left panel ). Middle and right panel: DNA yield of bisulfite-converted DNA as determined via UV ( middle panel ) measurement and via CFF qPCR assay ( right panel ), respectively. Shown are mean values of triplicate PCR measurements.

    Journal: PLoS ONE

    Article Title: Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

    doi: 10.1371/journal.pone.0093933

    Figure Lengend Snippet: DNA Yield and Purity after Bisulfite Conversion Applying Nine Different Kits. HMW ( upper panel ) and FFPE tissue ( lower panel ) DNA was bisulfite-converted using nine different commercially available kits. Each bisulfite conversion was conducted in nine replicates for each kit. Left panel: UV spectra of bisulfite-converted and purified DNA ( left panel ). Middle and right panel: DNA yield of bisulfite-converted DNA as determined via UV ( middle panel ) measurement and via CFF qPCR assay ( right panel ), respectively. Shown are mean values of triplicate PCR measurements.

    Article Snippet: Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits).

    Techniques: Formalin-fixed Paraffin-Embedded, Purification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in DNA from WT and DKO cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software 64 from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. 39 Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.

    Journal: Epigenetics

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    doi: 10.1080/15592294.2014.1003747

    Figure Lengend Snippet: Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in DNA from WT and DKO cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software 64 from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. 39 Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.

    Article Snippet: DNA (WT and DKO; 500 ng per sample) was bisulfite converted using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer's instructions.

    Techniques: Methylated DNA Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Magnetic Cell Separation, Software, Sequencing, Genome Wide, Methylation

    Validation of Ion Torrent-compatible MeDIP-Seq data by integration with the single-nucleotide resolution 450K array. ( a ) Histogram of the frequency and distribution of CpG cytosine methylation levels of probes on the 450K array in HCT116-WT (green) and -DKO (orange) cells stratified into categories based on their percentage of methylation. ( b ) Percentage of CpGs on the 450K array (black) overlapping with those underlying MeDIP-Seq peaks (white) of WT cells. ( c ) Distribution of methylation levels measured in WT cells of all probes on the 450K array that integrate with MeDIP-Seq peaks. ( d ) Distribution of methylation levels measured in DKO cells of all probes on the 450K array that integrate with MeDIP-Seq peaks in WT cells. ( e ) Scaled chromosomal view of the MeDIP-Seq enrichment of methylation over the ICR of the SNRPN gene. MeDIP-Seq from WT and DKO cells are displayed as indicated below the RefSeq annotation of SNRPN/SNURF (blue lines) and a CpG island (green). CpG methylation data of each 450K array probe over this region from WT and DKO cells are shown to scale as colored bars representing the degree of methylation (blue, low-; violet, intermediate-; and orange, high-levels of methylation). Percent methylation (calculated from β-values) of these CpGs is shown under each bar. The expanded region below these values shows a scaled depiction of CpG methylation of alleles sequenced after bisulfite-PCR in WT and DKO cells as indicated. Each circle represents a CpG cytosine in the amplicon sequenced, the color of which depicts its methylation state (white, unmethylated; black, methylated). *, the CpG on the 450K array also covered by the bisulfite-PCR amplicon. ( f ) The average expression of SNURF is significantly elevated in DKO compared to WT cells. Average expression values and s.e.m. from triplicate qPCR are shown. ( g ) DNA methylation over the SNURF promoter region is significantly higher in WT compared to DKO cells. Using bisulfite-PCR sequencing data described in e , percent methylation was calculated from the sum of methylated CpGs over the total CpGs evaluated. Average methylation values and s.e.m from individual clones are shown. P -values shown in f and g were calculated using 2-sided Student's t -test.

    Journal: Epigenetics

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    doi: 10.1080/15592294.2014.1003747

    Figure Lengend Snippet: Validation of Ion Torrent-compatible MeDIP-Seq data by integration with the single-nucleotide resolution 450K array. ( a ) Histogram of the frequency and distribution of CpG cytosine methylation levels of probes on the 450K array in HCT116-WT (green) and -DKO (orange) cells stratified into categories based on their percentage of methylation. ( b ) Percentage of CpGs on the 450K array (black) overlapping with those underlying MeDIP-Seq peaks (white) of WT cells. ( c ) Distribution of methylation levels measured in WT cells of all probes on the 450K array that integrate with MeDIP-Seq peaks. ( d ) Distribution of methylation levels measured in DKO cells of all probes on the 450K array that integrate with MeDIP-Seq peaks in WT cells. ( e ) Scaled chromosomal view of the MeDIP-Seq enrichment of methylation over the ICR of the SNRPN gene. MeDIP-Seq from WT and DKO cells are displayed as indicated below the RefSeq annotation of SNRPN/SNURF (blue lines) and a CpG island (green). CpG methylation data of each 450K array probe over this region from WT and DKO cells are shown to scale as colored bars representing the degree of methylation (blue, low-; violet, intermediate-; and orange, high-levels of methylation). Percent methylation (calculated from β-values) of these CpGs is shown under each bar. The expanded region below these values shows a scaled depiction of CpG methylation of alleles sequenced after bisulfite-PCR in WT and DKO cells as indicated. Each circle represents a CpG cytosine in the amplicon sequenced, the color of which depicts its methylation state (white, unmethylated; black, methylated). *, the CpG on the 450K array also covered by the bisulfite-PCR amplicon. ( f ) The average expression of SNURF is significantly elevated in DKO compared to WT cells. Average expression values and s.e.m. from triplicate qPCR are shown. ( g ) DNA methylation over the SNURF promoter region is significantly higher in WT compared to DKO cells. Using bisulfite-PCR sequencing data described in e , percent methylation was calculated from the sum of methylated CpGs over the total CpGs evaluated. Average methylation values and s.e.m from individual clones are shown. P -values shown in f and g were calculated using 2-sided Student's t -test.

    Article Snippet: DNA (WT and DKO; 500 ng per sample) was bisulfite converted using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer's instructions.

    Techniques: Methylated DNA Immunoprecipitation, Methylation, CpG Methylation Assay, Polymerase Chain Reaction, Amplification, Expressing, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Sequencing, Clone Assay

    Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.

    Journal: Epigenetics

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    doi: 10.1080/15592294.2014.1003747

    Figure Lengend Snippet: Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.

    Article Snippet: DNA (WT and DKO; 500 ng per sample) was bisulfite converted using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer's instructions.

    Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Genome Wide, Isolation, Sonication, Sequencing, Methylation, Immunoprecipitation, Agarose Gel Electrophoresis, Amplification, Concentration Assay, Chromatin Immunoprecipitation, Electron Microscopy, Software

    Ion Torrent-compatible MeDIP-Seq detects expected differences in DNA methylation between DNMT -proficient and - deficient cells. Distribution of 5mC methylation over the indicated bp window of all RefSeq annotated TSS ( a ), gene bodies ( b ), CpG islands ( c ), and exons ( d ). Methylation of WT (green line) and DKO (orange line) HCT116 cells are displayed as mean RPM values with s.e.m. indicated as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between WT and DKO methylation calculated using a one-sided KS test ( P -values shown). In c and d , a KS test was performed using data over the non-shaded areas as well ( P -values shown).

    Journal: Epigenetics

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    doi: 10.1080/15592294.2014.1003747

    Figure Lengend Snippet: Ion Torrent-compatible MeDIP-Seq detects expected differences in DNA methylation between DNMT -proficient and - deficient cells. Distribution of 5mC methylation over the indicated bp window of all RefSeq annotated TSS ( a ), gene bodies ( b ), CpG islands ( c ), and exons ( d ). Methylation of WT (green line) and DKO (orange line) HCT116 cells are displayed as mean RPM values with s.e.m. indicated as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between WT and DKO methylation calculated using a one-sided KS test ( P -values shown). In c and d , a KS test was performed using data over the non-shaded areas as well ( P -values shown).

    Article Snippet: DNA (WT and DKO; 500 ng per sample) was bisulfite converted using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer's instructions.

    Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation

    Ion Torrent-compatible MeDIP-Seq confirms a role for DNA methylation in alternative splicing. (a) Distribution of 5mC methylation over alternatively spliced exons (ASE) flanked by constitutively spliced exons (5’ or 3’ exons) in WT and DKO cells. Mean RPM values are displayed over ± 500 bp windows relative to the splice acceptor and donor sites at each of the exons as indicated, with s.e.m. depicted by shaded areas for WT (green) and DKO (orange) profiles. A schematic representation of cassette exons is shown, with the number of exons aberrantly included (blue) and excluded (gray) in WT vs. DKO cells indicated. P- values shown were calculated using one-sided KS test. (b) The ASE of the FAM204A gene (exon 2) is aberrantly excluded in DKO compared to WT cells. Schematic shows the 2 known isoforms for the FAM204A gene (blue bars depict the first 3 exons of the gene in the orientation indicated), under which a Sashimi plot generated by MISO analysis of RNA-Seq data measured as RPKM in WT and DKO cells illustrates the number of exon-exon junction reads as indicated to infer isoform expression. The left graphs show the MISO calculated distribution of a percent exon inclusion score (Psi-value; 95% confidence intervals in brackets) for the FAM204A ASE from WT (top) and DKO (bottom) RNA-Seq data. (c) Demethylation of the FAM204A ASE correlates with its aberrant exclusion. Scaled chromosomal view at the FAM204A gene region (ASE highlighted in yellow) of the indicated WT and DKO distribution of RNA-Seq (top pair) and DNA methylation (bottom pair) data displayed as RPKM. The first 3 exons of the gene are represented (blue bars) with a CpG island in the promoter region indicated (green bar). In the MeDIP-Seq data, red vertical lines correspond to areas containing CpGs.

    Journal: Epigenetics

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    doi: 10.1080/15592294.2014.1003747

    Figure Lengend Snippet: Ion Torrent-compatible MeDIP-Seq confirms a role for DNA methylation in alternative splicing. (a) Distribution of 5mC methylation over alternatively spliced exons (ASE) flanked by constitutively spliced exons (5’ or 3’ exons) in WT and DKO cells. Mean RPM values are displayed over ± 500 bp windows relative to the splice acceptor and donor sites at each of the exons as indicated, with s.e.m. depicted by shaded areas for WT (green) and DKO (orange) profiles. A schematic representation of cassette exons is shown, with the number of exons aberrantly included (blue) and excluded (gray) in WT vs. DKO cells indicated. P- values shown were calculated using one-sided KS test. (b) The ASE of the FAM204A gene (exon 2) is aberrantly excluded in DKO compared to WT cells. Schematic shows the 2 known isoforms for the FAM204A gene (blue bars depict the first 3 exons of the gene in the orientation indicated), under which a Sashimi plot generated by MISO analysis of RNA-Seq data measured as RPKM in WT and DKO cells illustrates the number of exon-exon junction reads as indicated to infer isoform expression. The left graphs show the MISO calculated distribution of a percent exon inclusion score (Psi-value; 95% confidence intervals in brackets) for the FAM204A ASE from WT (top) and DKO (bottom) RNA-Seq data. (c) Demethylation of the FAM204A ASE correlates with its aberrant exclusion. Scaled chromosomal view at the FAM204A gene region (ASE highlighted in yellow) of the indicated WT and DKO distribution of RNA-Seq (top pair) and DNA methylation (bottom pair) data displayed as RPKM. The first 3 exons of the gene are represented (blue bars) with a CpG island in the promoter region indicated (green bar). In the MeDIP-Seq data, red vertical lines correspond to areas containing CpGs.

    Article Snippet: DNA (WT and DKO; 500 ng per sample) was bisulfite converted using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer's instructions.

    Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation, Generated, RNA Sequencing Assay, Expressing

    Comparison of DNA methylation maps obtained with MeDIP, MethylCap, RRBS and Infinium DNA methylation maps were generated using MeDIP (first two tracks, in green), MethylCap (three tracks in blue, grey and red), RRBS (stacked blue tracks) and Infinium (single black track with percentage values) and converted into UCSC Genome Browser tracks. The screenshot shows the HOXA cluster in a human ES cell line (HUES6). Each track represents data from a single sequencing lane (MeDIP, MethylCap, RRBS) or microarray hybridization (Infinium). MeDIP and MethylCap data are visually similar to ChIP-seq data, with peaks in regions that exhibit high density of the target molecule (5-methyl-cytosine) and troughs in regions with low density of methylated cytosines. The height of the peaks represents the number of reads in each genomic interval, for each track normalized to the same genome-wide read count (note the twofold compressed scaling of the MethylCap tracks relative to the MeDIP tracks, which is indicative of higher dynamic range for MethylCap compared to MeDIP). RRBS gives rise to clusters of CpGs with absolute DNA methylation measurements, separated by regions that are not covered due to the reduced-representation property of the RRBS protocol. Each data point corresponds to the methylation level at a single CpG, and dark blue points indicate higher methylation levels than light blue points. Infinium data is represented in a similar way as the RRBS data, and the methylation levels at single CpGs are shown as percentage values. The three grey columns highlight regions that are illustrative of specific properties of the enrichment methods: (1) A promoter region that is CpG-poor and therefore not detectable by MeDIP or MethylCap – independent of its DNA methylation level; (2) a promoter region that contains many CpGs but low levels of DNA methylation, which also results in the absence of MeDIP and MethylCap peaks; and (3) a CpG island that exhibits a strong enrichment peak for both MeDIP and MethylCap although the RRBS data indicates that it is only partially methylated. For reference, the CpG density is indicated by stacked points (black) at the bottom of the diagram, and CpG islands (red) as well as known genes (blue) are listed as described previously 64 , 65 . All DNA methylation maps are available online as custom tracks for interactive visualization in the UCSC Genome Browser ( http://meth-benchmark.computational-epigenetics.org/ ).

    Journal: Nature biotechnology

    Article Title: Genome-wide mapping of DNA methylation: a quantitative technology comparison

    doi: 10.1038/nbt.1681

    Figure Lengend Snippet: Comparison of DNA methylation maps obtained with MeDIP, MethylCap, RRBS and Infinium DNA methylation maps were generated using MeDIP (first two tracks, in green), MethylCap (three tracks in blue, grey and red), RRBS (stacked blue tracks) and Infinium (single black track with percentage values) and converted into UCSC Genome Browser tracks. The screenshot shows the HOXA cluster in a human ES cell line (HUES6). Each track represents data from a single sequencing lane (MeDIP, MethylCap, RRBS) or microarray hybridization (Infinium). MeDIP and MethylCap data are visually similar to ChIP-seq data, with peaks in regions that exhibit high density of the target molecule (5-methyl-cytosine) and troughs in regions with low density of methylated cytosines. The height of the peaks represents the number of reads in each genomic interval, for each track normalized to the same genome-wide read count (note the twofold compressed scaling of the MethylCap tracks relative to the MeDIP tracks, which is indicative of higher dynamic range for MethylCap compared to MeDIP). RRBS gives rise to clusters of CpGs with absolute DNA methylation measurements, separated by regions that are not covered due to the reduced-representation property of the RRBS protocol. Each data point corresponds to the methylation level at a single CpG, and dark blue points indicate higher methylation levels than light blue points. Infinium data is represented in a similar way as the RRBS data, and the methylation levels at single CpGs are shown as percentage values. The three grey columns highlight regions that are illustrative of specific properties of the enrichment methods: (1) A promoter region that is CpG-poor and therefore not detectable by MeDIP or MethylCap – independent of its DNA methylation level; (2) a promoter region that contains many CpGs but low levels of DNA methylation, which also results in the absence of MeDIP and MethylCap peaks; and (3) a CpG island that exhibits a strong enrichment peak for both MeDIP and MethylCap although the RRBS data indicates that it is only partially methylated. For reference, the CpG density is indicated by stacked points (black) at the bottom of the diagram, and CpG islands (red) as well as known genes (blue) are listed as described previously 64 , 65 . All DNA methylation maps are available online as custom tracks for interactive visualization in the UCSC Genome Browser ( http://meth-benchmark.computational-epigenetics.org/ ).

    Article Snippet: MeDIP was performed using the EZ DNA methylation kit (Zymo Research).

    Techniques: DNA Methylation Assay, Methylated DNA Immunoprecipitation, Generated, Sequencing, Microarray, Hybridization, Chromatin Immunoprecipitation, Methylation, Genome Wide

    Detection of differentially methylated regions with MeDIP, MethylCap and RRBS Average DNA methylation measurements were calculated for each CpG island and compared between two human ES cell lines (HUES6 and HUES8). Total read frequencies are shown for MeDIP (panel A) and MethylCap (panel B), and mean DNA methylation levels are shown for RRBS (panel C). Regions with insufficient sequencing coverage were excluded. The Venn diagram (panel D) displays the total number and mutual overlap of differentially methylated CpG islands that could be identified by each method. CpG islands were classified as hypermethylated or hypomethylated (depending on the directionality of the difference) if the absolute DNA methylation difference exceeded 20% (for RRBS) or if there was at least a twofold difference in read number between the two samples (for MeDIP and MethylCap) – but only if Fisher’s exact test with multiple-testing correction gave rise to an estimated false-discovery rate of differential DNA methylation that was less than 0.1.

    Journal: Nature biotechnology

    Article Title: Genome-wide mapping of DNA methylation: a quantitative technology comparison

    doi: 10.1038/nbt.1681

    Figure Lengend Snippet: Detection of differentially methylated regions with MeDIP, MethylCap and RRBS Average DNA methylation measurements were calculated for each CpG island and compared between two human ES cell lines (HUES6 and HUES8). Total read frequencies are shown for MeDIP (panel A) and MethylCap (panel B), and mean DNA methylation levels are shown for RRBS (panel C). Regions with insufficient sequencing coverage were excluded. The Venn diagram (panel D) displays the total number and mutual overlap of differentially methylated CpG islands that could be identified by each method. CpG islands were classified as hypermethylated or hypomethylated (depending on the directionality of the difference) if the absolute DNA methylation difference exceeded 20% (for RRBS) or if there was at least a twofold difference in read number between the two samples (for MeDIP and MethylCap) – but only if Fisher’s exact test with multiple-testing correction gave rise to an estimated false-discovery rate of differential DNA methylation that was less than 0.1.

    Article Snippet: MeDIP was performed using the EZ DNA methylation kit (Zymo Research).

    Techniques: Methylation, Methylated DNA Immunoprecipitation, DNA Methylation Assay, Sequencing

    Quantification of DNA methylation with MeDIP, MethylCap and RRBS Absolute DNA methylation levels were calculated from the data obtained by MeDIP (panel A), MethylCap (panel B) and RRBS (panel C), respectively, and compared to DNA methylation levels determined by the Infinium assay. For MeDIP and MethylCap, sequencing reads were counted in 1-kilobase regions surrounding each CpG that is interrogated by the Infinium assay, and a regression model was used to infer absolute DNA methylation levels. Scatterplots and correlation coefficients were calculated on a test set that was not used for model fitting or feature selection. For RRBS, the DNA methylation level was determined as the percentage of methylated CpGs within 200 basepairs surrounding each CpG that is interrogated by the Infinium assay. Data shown are for the HUES6 human ES cell line, and regions that did not have sufficient sequencing coverage were excluded.

    Journal: Nature biotechnology

    Article Title: Genome-wide mapping of DNA methylation: a quantitative technology comparison

    doi: 10.1038/nbt.1681

    Figure Lengend Snippet: Quantification of DNA methylation with MeDIP, MethylCap and RRBS Absolute DNA methylation levels were calculated from the data obtained by MeDIP (panel A), MethylCap (panel B) and RRBS (panel C), respectively, and compared to DNA methylation levels determined by the Infinium assay. For MeDIP and MethylCap, sequencing reads were counted in 1-kilobase regions surrounding each CpG that is interrogated by the Infinium assay, and a regression model was used to infer absolute DNA methylation levels. Scatterplots and correlation coefficients were calculated on a test set that was not used for model fitting or feature selection. For RRBS, the DNA methylation level was determined as the percentage of methylated CpGs within 200 basepairs surrounding each CpG that is interrogated by the Infinium assay. Data shown are for the HUES6 human ES cell line, and regions that did not have sufficient sequencing coverage were excluded.

    Article Snippet: MeDIP was performed using the EZ DNA methylation kit (Zymo Research).

    Techniques: DNA Methylation Assay, Methylated DNA Immunoprecipitation, Sequencing, Selection, Methylation

    Genomic coverage of MeDIP, MethylCap, RRBS and Infinium Genomic coverage was quantified by the number of DNA methylation measurements that overlap with CpG islands (top row), gene promoters (center row) and a 1-kilobase tiling of the genome (bottom row). For MeDIP and MethylCap, the number of measurements is equal to the number of unique sequencing reads that fall inside each region. For RRBS, it refers to the number of valid DNA methylation measurements at CpGs within each region (one RRBS sequencing read typically yields one measurement, but can also give rise to more than one measurement if it contains several CpGs). For Infinium, the number of measurements is equal to the number of CpGs within each region that are present on the HumanMethylation27 microarray. CpG islands were calculated using CgiHunter ( http://cgihunter.bioinf.mpi-inf.mpg.de/ ), requiring a minimum CpG observed vs. expected ratio of 0.6, a minimum GC content of 0.5 and a minimum length of 700 basepairs 64 . Promoter regions were calculated based on Ensembl gene annotations, such that the region starts one kilo-base upstream of the annotated transcription start site (TSS) and extends to one kilobase downstream of the TSS. The genomic tiling was obtained by sliding a 1-kilobase window through the genome such that each tile starts at the position where the previous tile ends. No repeat-masking was performed for any of the three types of genomic regions. Data are shown for the HUES6 human ES cell line.

    Journal: Nature biotechnology

    Article Title: Genome-wide mapping of DNA methylation: a quantitative technology comparison

    doi: 10.1038/nbt.1681

    Figure Lengend Snippet: Genomic coverage of MeDIP, MethylCap, RRBS and Infinium Genomic coverage was quantified by the number of DNA methylation measurements that overlap with CpG islands (top row), gene promoters (center row) and a 1-kilobase tiling of the genome (bottom row). For MeDIP and MethylCap, the number of measurements is equal to the number of unique sequencing reads that fall inside each region. For RRBS, it refers to the number of valid DNA methylation measurements at CpGs within each region (one RRBS sequencing read typically yields one measurement, but can also give rise to more than one measurement if it contains several CpGs). For Infinium, the number of measurements is equal to the number of CpGs within each region that are present on the HumanMethylation27 microarray. CpG islands were calculated using CgiHunter ( http://cgihunter.bioinf.mpi-inf.mpg.de/ ), requiring a minimum CpG observed vs. expected ratio of 0.6, a minimum GC content of 0.5 and a minimum length of 700 basepairs 64 . Promoter regions were calculated based on Ensembl gene annotations, such that the region starts one kilo-base upstream of the annotated transcription start site (TSS) and extends to one kilobase downstream of the TSS. The genomic tiling was obtained by sliding a 1-kilobase window through the genome such that each tile starts at the position where the previous tile ends. No repeat-masking was performed for any of the three types of genomic regions. Data are shown for the HUES6 human ES cell line.

    Article Snippet: MeDIP was performed using the EZ DNA methylation kit (Zymo Research).

    Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Sequencing, Microarray, Gas Chromatography

    DNA integrity and size range as assessed by agarose gel electrophoresis. (A) DNA size markers used to estimate size ranges are shown in addition to genomic DNA that was isolated from white blood cells (WBC) and used as a positive control. (B) Representative DBS from each of the tested protocols are shown, except for protocol QQ in which DNA amounts were insufficient to be analyzed by gel electrophoresis. Eight different gel sections are shown and are derived from either the same gel or different gels. In each section, two punches from the same NCS spot were run on the same gel, with the first punch, labeled ‘a’, representing protocol GQ and the second punch, labeled ‘b’, representing another unique protocol from the tested set. The two blue lines, representing the 100 and 1000 base pair (bp) size ranges, were set according to the molecular size marker used in each section. The 1000 base pair limit is a minimum size range with useful applications in many genetic and epigenetic studies, including Illumina’s HM450 Beadchip array. The results of other DBS analyzed by gel electrophoresis or bioanalyzer are summarized in Table 6 .

    Journal: BMC Biotechnology

    Article Title: Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

    doi: 10.1186/1472-6750-14-60

    Figure Lengend Snippet: DNA integrity and size range as assessed by agarose gel electrophoresis. (A) DNA size markers used to estimate size ranges are shown in addition to genomic DNA that was isolated from white blood cells (WBC) and used as a positive control. (B) Representative DBS from each of the tested protocols are shown, except for protocol QQ in which DNA amounts were insufficient to be analyzed by gel electrophoresis. Eight different gel sections are shown and are derived from either the same gel or different gels. In each section, two punches from the same NCS spot were run on the same gel, with the first punch, labeled ‘a’, representing protocol GQ and the second punch, labeled ‘b’, representing another unique protocol from the tested set. The two blue lines, representing the 100 and 1000 base pair (bp) size ranges, were set according to the molecular size marker used in each section. The 1000 base pair limit is a minimum size range with useful applications in many genetic and epigenetic studies, including Illumina’s HM450 Beadchip array. The results of other DBS analyzed by gel electrophoresis or bioanalyzer are summarized in Table 6 .

    Article Snippet: DBS DNA (300 ng) samples were bisulfite converted using EZ DNA Methylation Kit (Zymo Research D5001) according to manufacturer’s instructions.

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Isolation, Positive Control, Nucleic Acid Electrophoresis, Derivative Assay, Labeling, Marker

    Phases and classification of protocols used to extract DNA from DBS. Two sequential phases, each encompassing three steps, are outlined (A) and were optimized in the different protocols or method combinations (B) used to extract DNA from DBS. A spin basket is shown next to Phase I.A and consists of a tube with an embedded perforated basket used to separate blood solutions from the filter papers from which they were extracted. A silica-gel column with a funnel-shape design is shown next to Phase II.E and often used to elute small volumes (5–30 μl), as manufactured by Macherey-Nagel and supplied with the extra-small (XS) versions of NucleoSpin kits (B) .

    Journal: BMC Biotechnology

    Article Title: Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

    doi: 10.1186/1472-6750-14-60

    Figure Lengend Snippet: Phases and classification of protocols used to extract DNA from DBS. Two sequential phases, each encompassing three steps, are outlined (A) and were optimized in the different protocols or method combinations (B) used to extract DNA from DBS. A spin basket is shown next to Phase I.A and consists of a tube with an embedded perforated basket used to separate blood solutions from the filter papers from which they were extracted. A silica-gel column with a funnel-shape design is shown next to Phase II.E and often used to elute small volumes (5–30 μl), as manufactured by Macherey-Nagel and supplied with the extra-small (XS) versions of NucleoSpin kits (B) .

    Article Snippet: DBS DNA (300 ng) samples were bisulfite converted using EZ DNA Methylation Kit (Zymo Research D5001) according to manufacturer’s instructions.

    Techniques:

    HM450 QC plot using Non-polymorphic probes which assess overall performance. In the green channel, background signals are shown in red and pink while positive signals in opaque and fluorescent green. In the red channel, background signals are shown in opaque and fluorescent green while positive signals in red and pink. One non-polymorphic control has been designed for each of the four nucleotides A, T, C, and G. Four DBS DNA samples are shown between four neonatal blood and four cell line DNA samples, in each of the two plots. The DBS samples represent two NCS spots, 37 and 38, each consisting of two tested punches labeled ‘a’ or ‘b’.

    Journal: BMC Biotechnology

    Article Title: Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

    doi: 10.1186/1472-6750-14-60

    Figure Lengend Snippet: HM450 QC plot using Non-polymorphic probes which assess overall performance. In the green channel, background signals are shown in red and pink while positive signals in opaque and fluorescent green. In the red channel, background signals are shown in opaque and fluorescent green while positive signals in red and pink. One non-polymorphic control has been designed for each of the four nucleotides A, T, C, and G. Four DBS DNA samples are shown between four neonatal blood and four cell line DNA samples, in each of the two plots. The DBS samples represent two NCS spots, 37 and 38, each consisting of two tested punches labeled ‘a’ or ‘b’.

    Article Snippet: DBS DNA (300 ng) samples were bisulfite converted using EZ DNA Methylation Kit (Zymo Research D5001) according to manufacturer’s instructions.

    Techniques: Labeling

    Differential methylation and unsupervised clustering analysis of HM450 data from neonatal blood, DBS and cell line DNA. Neonatal blood and cell line DNA samples are used as positive controls of good DNA quality for reference comparisons with DNA extracted from DBS. Neonatal blood and DBS are from different individuals. Four DBS DNA samples are shown between four different neonatal blood and four different cell line DNA samples. The DBS samples represent two NCS spots, 37 and 38, each consisting of two tested punches labeled ‘a’ or ‘b’. HM450 beta-values were clustered using Euclidean distance as the dissimilarity index. As shown in the color key, the red and blue signals represent relatively hypomethylated and hypermethylated regions, respectively.

    Journal: BMC Biotechnology

    Article Title: Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

    doi: 10.1186/1472-6750-14-60

    Figure Lengend Snippet: Differential methylation and unsupervised clustering analysis of HM450 data from neonatal blood, DBS and cell line DNA. Neonatal blood and cell line DNA samples are used as positive controls of good DNA quality for reference comparisons with DNA extracted from DBS. Neonatal blood and DBS are from different individuals. Four DBS DNA samples are shown between four different neonatal blood and four different cell line DNA samples. The DBS samples represent two NCS spots, 37 and 38, each consisting of two tested punches labeled ‘a’ or ‘b’. HM450 beta-values were clustered using Euclidean distance as the dissimilarity index. As shown in the color key, the red and blue signals represent relatively hypomethylated and hypermethylated regions, respectively.

    Article Snippet: DBS DNA (300 ng) samples were bisulfite converted using EZ DNA Methylation Kit (Zymo Research D5001) according to manufacturer’s instructions.

    Techniques: Methylation, Labeling

    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Article Snippet: The ‘post-bisulphite’ conversion WGBS libraries were prepared according to the Accel-NGS Methyl-Seq DNA library kit (Swift BioSciences) or the TruSeq DNA Methylation Kit (Illumina) according to the manufacturers’ protocols.

    Techniques: Bisulfite Sequencing, Methylation, Construct, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, DNA Methylation Assay, Random Hexamer Labeling, DNA Ligation, Modification, Ligation

    Sequence bias in whole genome bisulphite sequencing libraries. ( A ) GC bias observed for the different methods. The plots show the normalised coverage in 100 bp windows of increasing GC content in the human reference genome. NEBNextUltra libraries had higher read coverage in AT rich regions, whilst TruSeq DNA Methylation had increased read coverage of GC rich regions. The AccelNGS Methyl-Seq and SPLAT libraries displayed a lower GC bias, however regions with extreme GC content were not well represented. ( B ) Cumulative coverage of different genomic regions. Coverage plots for the whole genome is shown in in green, CpG islands in purple, FANTOM5 enhancer regions in blue and a set of 1000 promoter regions that are difficult to sequence due to high GC content in grey.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Sequence bias in whole genome bisulphite sequencing libraries. ( A ) GC bias observed for the different methods. The plots show the normalised coverage in 100 bp windows of increasing GC content in the human reference genome. NEBNextUltra libraries had higher read coverage in AT rich regions, whilst TruSeq DNA Methylation had increased read coverage of GC rich regions. The AccelNGS Methyl-Seq and SPLAT libraries displayed a lower GC bias, however regions with extreme GC content were not well represented. ( B ) Cumulative coverage of different genomic regions. Coverage plots for the whole genome is shown in in green, CpG islands in purple, FANTOM5 enhancer regions in blue and a set of 1000 promoter regions that are difficult to sequence due to high GC content in grey.

    Article Snippet: The ‘post-bisulphite’ conversion WGBS libraries were prepared according to the Accel-NGS Methyl-Seq DNA library kit (Swift BioSciences) or the TruSeq DNA Methylation Kit (Illumina) according to the manufacturers’ protocols.

    Techniques: Sequencing, Bisulfite Sequencing, Gas Chromatography, DNA Methylation Assay

    Comparison of SPLAT with a high coverage reference dataset. ( A ) Pairwise comparison of methylation levels at individual CpG sites in CpG islands, enhancers, and repetitive regions determined by SPLAT (x-axis) and the high-coverage reference WGBS data (y-axis) obtained by combining the data from TruSeq DNA Methylation, Accel-NGS and NEBNextUltra libraries. Pearson's correlation coefficients are shown in the lower right corner of each scatter plot. ( B ) The mean read coverage of the CpG sites in CpG islands, enhancers and repeats for SPLAT and the high coverage reference data set. ( C ) Comparison of the numbers and overlaps of un-methylated regions (UMRs) between SPLAT and high coverage reference WGBS data. ( D ) Comparison of the numbers and overlaps of lowly methylated regions (LMRs) in SPLAT and high coverage reference WGBS data.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Comparison of SPLAT with a high coverage reference dataset. ( A ) Pairwise comparison of methylation levels at individual CpG sites in CpG islands, enhancers, and repetitive regions determined by SPLAT (x-axis) and the high-coverage reference WGBS data (y-axis) obtained by combining the data from TruSeq DNA Methylation, Accel-NGS and NEBNextUltra libraries. Pearson's correlation coefficients are shown in the lower right corner of each scatter plot. ( B ) The mean read coverage of the CpG sites in CpG islands, enhancers and repeats for SPLAT and the high coverage reference data set. ( C ) Comparison of the numbers and overlaps of un-methylated regions (UMRs) between SPLAT and high coverage reference WGBS data. ( D ) Comparison of the numbers and overlaps of lowly methylated regions (LMRs) in SPLAT and high coverage reference WGBS data.

    Article Snippet: The ‘post-bisulphite’ conversion WGBS libraries were prepared according to the Accel-NGS Methyl-Seq DNA library kit (Swift BioSciences) or the TruSeq DNA Methylation Kit (Illumina) according to the manufacturers’ protocols.

    Techniques: Methylation, DNA Methylation Assay, Next-Generation Sequencing

    Annotation of CpG sites with low coverage in the ‘post bisulphite’ WGBS methods. A coverage threshold of ≤2 reads was applied to identify CpG sites that were insufficiently represented by the different WGBS library methods (based on down-sampled data). In TruSeq DNA Methylation libraries, low coverage sites were mostly found in intergenic regions and introns. The SPLAT and Accel-NGS Methyl-Seq data displayed overall lower number of CpG sites with low coverage, although the number of poorly represented sites in CpG islands and promoter regions were higher compared to the TruSeq DNA methylation libraries.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Annotation of CpG sites with low coverage in the ‘post bisulphite’ WGBS methods. A coverage threshold of ≤2 reads was applied to identify CpG sites that were insufficiently represented by the different WGBS library methods (based on down-sampled data). In TruSeq DNA Methylation libraries, low coverage sites were mostly found in intergenic regions and introns. The SPLAT and Accel-NGS Methyl-Seq data displayed overall lower number of CpG sites with low coverage, although the number of poorly represented sites in CpG islands and promoter regions were higher compared to the TruSeq DNA methylation libraries.

    Article Snippet: The ‘post-bisulphite’ conversion WGBS libraries were prepared according to the Accel-NGS Methyl-Seq DNA library kit (Swift BioSciences) or the TruSeq DNA Methylation Kit (Illumina) according to the manufacturers’ protocols.

    Techniques: DNA Methylation Assay, Next-Generation Sequencing

    AC with hypomethylated DNA are unable to suppress inflammatory arthritis. DC were left untreated and made apoptotic (AC) or treated with 5-azacytidine and then made apoptotic (AZA AC) and the level of global methylation analyzed by ELISA ( A ). Mice were left untreated (UT) or injected with AC or AZA AC (AZA) at the time of immunization with CFA/mBSA and for a further 2 consecutive days. Arthritis was induced 7 days later and clinical score assessed for a further 7 days ( B ) and knee swelling determined 3 days post knee injections ( C ). 7 days post knee injection, draining lymph nodes were harvested and analyzed for IL-17 production by ELISA ( D ). Graphs shows mean cytokine production ± SEM. Data is pooled from 4 independent experiments with a total of 12 mice per group. Statistical analysis was performed using a t-test ( A , paired; C and D , unpaired) or a one-way analysis of variance (ANOVA) followed by Bonferroni post hoc testing for multiple comparisons ( B ).

    Journal: Scientific Reports

    Article Title: DNA methylation governs the dynamic regulation of inflammation by apoptotic cells during efferocytosis

    doi: 10.1038/srep42204

    Figure Lengend Snippet: AC with hypomethylated DNA are unable to suppress inflammatory arthritis. DC were left untreated and made apoptotic (AC) or treated with 5-azacytidine and then made apoptotic (AZA AC) and the level of global methylation analyzed by ELISA ( A ). Mice were left untreated (UT) or injected with AC or AZA AC (AZA) at the time of immunization with CFA/mBSA and for a further 2 consecutive days. Arthritis was induced 7 days later and clinical score assessed for a further 7 days ( B ) and knee swelling determined 3 days post knee injections ( C ). 7 days post knee injection, draining lymph nodes were harvested and analyzed for IL-17 production by ELISA ( D ). Graphs shows mean cytokine production ± SEM. Data is pooled from 4 independent experiments with a total of 12 mice per group. Statistical analysis was performed using a t-test ( A , paired; C and D , unpaired) or a one-way analysis of variance (ANOVA) followed by Bonferroni post hoc testing for multiple comparisons ( B ).

    Article Snippet: The amount of DNA methylation was determined by measurement of the levels of 5-methyl-2-deoxy cytidine (ng/ml) using the DNA methylation ELISA kit (Cayman Chemical Company, MI, USA) following manufacturer’s instructions.

    Techniques: Methylation, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    Remethylation of DNA isolated from activated AC restores the ability of the DNA to suppress inflammatory arthritis. ( A ) DNA was isolated from resting or activated AC and loaded into PE-labeled, cationic liposomes. Mice were intravenously injected with liposomes alone or liposomes containing resting AC (LAC), or activated AC DNA (LaAC) and spleens harvested 1 hr later. PE + cells were analyzed for co-staining with F4/80 (macrophages) and CD11c (DC). ( B ) DNA was isolated from resting AC, loaded into liposomes and injected into mice at the time of immunization with CFA/mBSA and for a further two consecutive days. Comparison was made with AC injected into mice at the same time points. Arthritis was induced 7 days later and clinical score assessed for 3 days. Data is pooled from 3 independent experiments, with a total of 8 mice/group. DNA was isolated from activated AC and left untreated (LaAC) or re-methylated with CpG methylase (LmaAC) and loaded into liposomes. ( C ) Prior to liposome loading, global DNA methylation was determined by ELISA. ( D ) Mice were left untreated (UT) or injected with liposomes alone, LaAC or LmaAC at the time of immunization with CFA/mBSA and for a further 2 consecutive days. Arthritis was induced 7 days later and clinical score assessed for a further 7 days. ( E ) 7 days post knee injection, draining lymph nodes were harvested and analyzed for IL-17 production by ELISA. Graph shows mean cytokine production ± SEM. Data is pooled from 4 independent experiments with a total of 12 mice per group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc testing for multiple comparisons ( B , D ) or a t-test ( E ).

    Journal: Scientific Reports

    Article Title: DNA methylation governs the dynamic regulation of inflammation by apoptotic cells during efferocytosis

    doi: 10.1038/srep42204

    Figure Lengend Snippet: Remethylation of DNA isolated from activated AC restores the ability of the DNA to suppress inflammatory arthritis. ( A ) DNA was isolated from resting or activated AC and loaded into PE-labeled, cationic liposomes. Mice were intravenously injected with liposomes alone or liposomes containing resting AC (LAC), or activated AC DNA (LaAC) and spleens harvested 1 hr later. PE + cells were analyzed for co-staining with F4/80 (macrophages) and CD11c (DC). ( B ) DNA was isolated from resting AC, loaded into liposomes and injected into mice at the time of immunization with CFA/mBSA and for a further two consecutive days. Comparison was made with AC injected into mice at the same time points. Arthritis was induced 7 days later and clinical score assessed for 3 days. Data is pooled from 3 independent experiments, with a total of 8 mice/group. DNA was isolated from activated AC and left untreated (LaAC) or re-methylated with CpG methylase (LmaAC) and loaded into liposomes. ( C ) Prior to liposome loading, global DNA methylation was determined by ELISA. ( D ) Mice were left untreated (UT) or injected with liposomes alone, LaAC or LmaAC at the time of immunization with CFA/mBSA and for a further 2 consecutive days. Arthritis was induced 7 days later and clinical score assessed for a further 7 days. ( E ) 7 days post knee injection, draining lymph nodes were harvested and analyzed for IL-17 production by ELISA. Graph shows mean cytokine production ± SEM. Data is pooled from 4 independent experiments with a total of 12 mice per group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc testing for multiple comparisons ( B , D ) or a t-test ( E ).

    Article Snippet: The amount of DNA methylation was determined by measurement of the levels of 5-methyl-2-deoxy cytidine (ng/ml) using the DNA methylation ELISA kit (Cayman Chemical Company, MI, USA) following manufacturer’s instructions.

    Techniques: Isolation, Labeling, Mouse Assay, Injection, Staining, Methylation, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay

    DNA methylation status of human AC is associated with pro- versus anti-inflammatory responses. ( A ) CD4 + T cells were isolated from PBMC of healthy controls and patients with RA and SLE and cultured overnight with etoposide to induce apoptosis. DNA was isolated from CD4 + AC and digested for the determination of global methylated cytidine levels by ELISA. Graphs show mean ± SEM. Healthy n = 6, RA n = 8, SLE n = 12. ( B , C ) The AC generated in ( A ) were cultured with monocyte-derived macrophages from healthy donors for 72 hours. Supernatants were assayed by ELISA for TGFβ and CBA for IL-6. ( D ) Ratio of IL-6 to TGFβ production by monocyte-derived macrophages. Graphs show mean cytokine production ± SEM. Statistical analysis was performed using a t-test.

    Journal: Scientific Reports

    Article Title: DNA methylation governs the dynamic regulation of inflammation by apoptotic cells during efferocytosis

    doi: 10.1038/srep42204

    Figure Lengend Snippet: DNA methylation status of human AC is associated with pro- versus anti-inflammatory responses. ( A ) CD4 + T cells were isolated from PBMC of healthy controls and patients with RA and SLE and cultured overnight with etoposide to induce apoptosis. DNA was isolated from CD4 + AC and digested for the determination of global methylated cytidine levels by ELISA. Graphs show mean ± SEM. Healthy n = 6, RA n = 8, SLE n = 12. ( B , C ) The AC generated in ( A ) were cultured with monocyte-derived macrophages from healthy donors for 72 hours. Supernatants were assayed by ELISA for TGFβ and CBA for IL-6. ( D ) Ratio of IL-6 to TGFβ production by monocyte-derived macrophages. Graphs show mean cytokine production ± SEM. Statistical analysis was performed using a t-test.

    Article Snippet: The amount of DNA methylation was determined by measurement of the levels of 5-methyl-2-deoxy cytidine (ng/ml) using the DNA methylation ELISA kit (Cayman Chemical Company, MI, USA) following manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Isolation, Cell Culture, Methylation, Enzyme-linked Immunosorbent Assay, Generated, Derivative Assay, Crocin Bleaching Assay

    The DNA from LPS-activated AC is hypomethylated compared to unstimulated AC. Bone marrow derived DC were either left unstimulated or activated with LPS overnight followed by induction of apoptosis by addition of etoposide for 5 hr. DNA was isolated from unstimulated apoptotic cells (AC) or activated apoptotic cells (aAC) and global methylation analyzed by ELISA ( A ) or HPLC ( B ). Statistical analysis was performed using an unpaired ( A ) or paired ( B ) t-test.

    Journal: Scientific Reports

    Article Title: DNA methylation governs the dynamic regulation of inflammation by apoptotic cells during efferocytosis

    doi: 10.1038/srep42204

    Figure Lengend Snippet: The DNA from LPS-activated AC is hypomethylated compared to unstimulated AC. Bone marrow derived DC were either left unstimulated or activated with LPS overnight followed by induction of apoptosis by addition of etoposide for 5 hr. DNA was isolated from unstimulated apoptotic cells (AC) or activated apoptotic cells (aAC) and global methylation analyzed by ELISA ( A ) or HPLC ( B ). Statistical analysis was performed using an unpaired ( A ) or paired ( B ) t-test.

    Article Snippet: The amount of DNA methylation was determined by measurement of the levels of 5-methyl-2-deoxy cytidine (ng/ml) using the DNA methylation ELISA kit (Cayman Chemical Company, MI, USA) following manufacturer’s instructions.

    Techniques: Derivative Assay, Isolation, Methylation, Enzyme-linked Immunosorbent Assay, High Performance Liquid Chromatography

    MALDI-TOF-MS DNA methylation analysis. Overview of the MassCLEAVE™ assay. (A) Genomic DNA is bisulphite treated and PCR-tagged to include the T7 promoter sequence. As shown, either top or bottom strand can be used for amplification. Subsequent alkaline phosphatase (SAP) treatment, in vitro transcription using T7 R DNA polymerase and a specific nucleotide mixture plus RNase A cleavage results in specific fragmentation. As exemplified, the top and bottom strands can have markedly different fragmentation patterns. The obtained mixture of fragments can be analyzed by MALDI-TOF-MS. Spectrum peaks representing methylated and unmethylated fragments are used to calculate methylation levels for every fragment. (B) In silico transcript fragmentation. The use of T- or C-cleavage mixtures on either the top or bottom strand of bisulphite-treated DNA can yield quite different fragmentation patterns. Here, the fragmentation of the CpG island of INHBB (chr2:119,998,230-119,998,596) is shown. CpG sites are represented by circles; white circle: methylation call will be obtained in the MassCLEAVE™ assay; crossed gray circle: methylation call will be missed, because fragments mass falls outside of spectral range of analysis; red diagonal line: approximate cleavage site; dotted line: combined methylation call due to overlapping peaks in spectrum. T-cleavage is more informative than C-cleavage when interrogating CpG islands due to RNaseA digestion after every CpG site in the C-cleavage reaction. (C) New schematic representation of DNA methylation data. Due to the specific fragmentation of a transcript, multiple CpG sites can be present within one fragment. Also, fragments with identical masses will show overlapping peaks in the MALDI-TOF-MS spectrum, resulting in a combined DNA methylation call. This new graphical representation of the DNA methylation incorporates all this information. The white circles represent the CpG sites that are analyzed in the MassCLEAVE™ assay. The crossed grey circles represent CpG sites that will be missed in the assay, and the red diagonal lines are the RNase A cleavage sites. The colored circles in the average view indicate the methylation calls given by the assay with the dashed lines linking these calls back to the CpG site(s) within the interrogated sequence. In the detailed view, the relative abundance of unmethylated, partially methylated, and fully methylated molecules is visualized as proportional bars for each fragment (white, gray and black bars, respectively). This view allows an in-depth graphical comparison of samples at the highest resolution possible.

    Journal: Nucleic Acids Research

    Article Title: Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements

    doi: 10.1093/nar/gkm662

    Figure Lengend Snippet: MALDI-TOF-MS DNA methylation analysis. Overview of the MassCLEAVE™ assay. (A) Genomic DNA is bisulphite treated and PCR-tagged to include the T7 promoter sequence. As shown, either top or bottom strand can be used for amplification. Subsequent alkaline phosphatase (SAP) treatment, in vitro transcription using T7 R DNA polymerase and a specific nucleotide mixture plus RNase A cleavage results in specific fragmentation. As exemplified, the top and bottom strands can have markedly different fragmentation patterns. The obtained mixture of fragments can be analyzed by MALDI-TOF-MS. Spectrum peaks representing methylated and unmethylated fragments are used to calculate methylation levels for every fragment. (B) In silico transcript fragmentation. The use of T- or C-cleavage mixtures on either the top or bottom strand of bisulphite-treated DNA can yield quite different fragmentation patterns. Here, the fragmentation of the CpG island of INHBB (chr2:119,998,230-119,998,596) is shown. CpG sites are represented by circles; white circle: methylation call will be obtained in the MassCLEAVE™ assay; crossed gray circle: methylation call will be missed, because fragments mass falls outside of spectral range of analysis; red diagonal line: approximate cleavage site; dotted line: combined methylation call due to overlapping peaks in spectrum. T-cleavage is more informative than C-cleavage when interrogating CpG islands due to RNaseA digestion after every CpG site in the C-cleavage reaction. (C) New schematic representation of DNA methylation data. Due to the specific fragmentation of a transcript, multiple CpG sites can be present within one fragment. Also, fragments with identical masses will show overlapping peaks in the MALDI-TOF-MS spectrum, resulting in a combined DNA methylation call. This new graphical representation of the DNA methylation incorporates all this information. The white circles represent the CpG sites that are analyzed in the MassCLEAVE™ assay. The crossed grey circles represent CpG sites that will be missed in the assay, and the red diagonal lines are the RNase A cleavage sites. The colored circles in the average view indicate the methylation calls given by the assay with the dashed lines linking these calls back to the CpG site(s) within the interrogated sequence. In the detailed view, the relative abundance of unmethylated, partially methylated, and fully methylated molecules is visualized as proportional bars for each fragment (white, gray and black bars, respectively). This view allows an in-depth graphical comparison of samples at the highest resolution possible.

    Article Snippet: A crucial step in the Sequenom® DNA methylation analysis is the PCR amplification, as a successful PCR is likely to result in a good MALDI-TOF-MS spectrum and thus in an accurate determination of the DNA methylation status of the fragments.

    Techniques: Mass Spectrometry, DNA Methylation Assay, Polymerase Chain Reaction, Sequencing, Amplification, In Vitro, Methylation, In Silico

    Variability in DNA methylation quantitation. Methylation variability due to bisulphite conversion or PCR amplification was evaluated. (A) In three independent experiments, a mixture of 25% enzymatically methylated gDNA and 75% blood gDNA (generally unmethylated) was bisulphite treated in triplicate and EN1 CpG island PCR-amplified in triplicate. The mean + SEM results are graphed per bisulphite conversion (BSC) and per fragment. The numbers below the graph indicate which CpG sites are interrogated together in the respective fragment and the numbering of the sites is according to the position in amplicon. The point (.) between numbers indicates adjacent CpG sites present within one fragments, whereas the equals to sign (=) is indicative of CpG sites that are part of different fragments, but analyzed together because of identical fragment masses. The variability in the methylation calls shown in this graph is caused by PCR amplification of bisulphite-treated single-stranded DNA. (B) To be able to interrogate the variability introduced by the bisulphite conversion, we calculated the average methylation calls of the triplicate PCR reactions on the same bisulphite-treated gDNA, and used these values to graph the DNA methylation calls per fragment obtained from three independent bisulphite conversions. Similar variability studies for SCTR and INHBB CpG island regions are given as Supplementary Data files.

    Journal: Nucleic Acids Research

    Article Title: Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements

    doi: 10.1093/nar/gkm662

    Figure Lengend Snippet: Variability in DNA methylation quantitation. Methylation variability due to bisulphite conversion or PCR amplification was evaluated. (A) In three independent experiments, a mixture of 25% enzymatically methylated gDNA and 75% blood gDNA (generally unmethylated) was bisulphite treated in triplicate and EN1 CpG island PCR-amplified in triplicate. The mean + SEM results are graphed per bisulphite conversion (BSC) and per fragment. The numbers below the graph indicate which CpG sites are interrogated together in the respective fragment and the numbering of the sites is according to the position in amplicon. The point (.) between numbers indicates adjacent CpG sites present within one fragments, whereas the equals to sign (=) is indicative of CpG sites that are part of different fragments, but analyzed together because of identical fragment masses. The variability in the methylation calls shown in this graph is caused by PCR amplification of bisulphite-treated single-stranded DNA. (B) To be able to interrogate the variability introduced by the bisulphite conversion, we calculated the average methylation calls of the triplicate PCR reactions on the same bisulphite-treated gDNA, and used these values to graph the DNA methylation calls per fragment obtained from three independent bisulphite conversions. Similar variability studies for SCTR and INHBB CpG island regions are given as Supplementary Data files.

    Article Snippet: A crucial step in the Sequenom® DNA methylation analysis is the PCR amplification, as a successful PCR is likely to result in a good MALDI-TOF-MS spectrum and thus in an accurate determination of the DNA methylation status of the fragments.

    Techniques: DNA Methylation Assay, Quantitation Assay, Methylation, Polymerase Chain Reaction, Amplification

    Template dilution test for transcription and subsequent MALDI-TOF analysis. The minimal amount of PCR template required for accurate quantitation of DNA methylation was determined. (A) A twofold dilution series was prepared from T7-tagged PCR amplifications of EN1 CpG island (50/50% mixture of fully unmethylated and fully methylated PCR product), ranging from 80 to 2.5 nM, and 5 μl of each dilution was visualized on an agarose gel. Adjacent marker lanes contain 500 ng pBR322 digested with Hin FI. (B) Two microliters of the dilution series was used in the transcription-RNase A digestion reaction and subjected to MALDI-TOF MS analysis, in five technical replicates. The ratio of detected versus expected fragments with CpG sites was determined in all spectra and are presented as individual bars for each analysis. (C) The average methylation calls ratios calculated from the detected peaks in the spectra are shown as means + SEM. The dotted horizontal line represents the actual methylation level used as input. Similar template dilution studies for SCTR and INHBB CpG island regions are given as Supplementary Data files.

    Journal: Nucleic Acids Research

    Article Title: Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements

    doi: 10.1093/nar/gkm662

    Figure Lengend Snippet: Template dilution test for transcription and subsequent MALDI-TOF analysis. The minimal amount of PCR template required for accurate quantitation of DNA methylation was determined. (A) A twofold dilution series was prepared from T7-tagged PCR amplifications of EN1 CpG island (50/50% mixture of fully unmethylated and fully methylated PCR product), ranging from 80 to 2.5 nM, and 5 μl of each dilution was visualized on an agarose gel. Adjacent marker lanes contain 500 ng pBR322 digested with Hin FI. (B) Two microliters of the dilution series was used in the transcription-RNase A digestion reaction and subjected to MALDI-TOF MS analysis, in five technical replicates. The ratio of detected versus expected fragments with CpG sites was determined in all spectra and are presented as individual bars for each analysis. (C) The average methylation calls ratios calculated from the detected peaks in the spectra are shown as means + SEM. The dotted horizontal line represents the actual methylation level used as input. Similar template dilution studies for SCTR and INHBB CpG island regions are given as Supplementary Data files.

    Article Snippet: A crucial step in the Sequenom® DNA methylation analysis is the PCR amplification, as a successful PCR is likely to result in a good MALDI-TOF-MS spectrum and thus in an accurate determination of the DNA methylation status of the fragments.

    Techniques: Polymerase Chain Reaction, Quantitation Assay, DNA Methylation Assay, Methylation, Agarose Gel Electrophoresis, Marker, Mass Spectrometry

    Accuracy and precision of MassCLEAVE™ DNA methylation detection in EN1 CpG island. The reproducibility and quantitation of different CpG methylation levels were assessed using MassCLEAVE™ technology (A) We measured the DNA methylation calls according to the MassCLEAVE™ technology, but using ASF (Supplementary Data). Known input ratios of completely methylated and unmethylated DNA fragments (referred to as 0, 5, 10, 25, 50, 75, 90 or 100% methylated input DNA; panels from upper left to lower right, respectively) were either analyzed immediately in a T7 transcription-RNase A digestion reaction, followed by a MALDI-TOF MS analysis (direct analysis, gray bars), or analyzed following an extra PCR step (PCR + analysis, black bars). For each panel, the methylation calls are arranged per fragment in an increasing mass order ( x -axis) and the measured methylation ratios ( y -axis) are given as means + SEM of five replicate measurements. The dotted vertical line indicates the 1700 Da threshold, and the horizontal dotted lines represent the input methylation levels. (B) Accuracy of DNA methylation calls by MassCLEAVE™ and PCR-induced bias. The left panel shows the mean DNA methylation calls of fragments with a mass over 1700 Da ( y -axis) plotted against the input levels ( x -axis), revealing a high correlation between input and output levels. The right panel shows the difference between the assay output and the known input (error) versus the input DNA methylation levels, revealing a small but consistent bias. The data for the direct analysis is plotted as a grey line and the data for PCR + analysis is shown as a dashed black line. Data are given as means + SEM of five replicate measurements. Similar accuracy and precision data for SCTR and INHBB CpG islands are given as Supplementary Data files.

    Journal: Nucleic Acids Research

    Article Title: Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements

    doi: 10.1093/nar/gkm662

    Figure Lengend Snippet: Accuracy and precision of MassCLEAVE™ DNA methylation detection in EN1 CpG island. The reproducibility and quantitation of different CpG methylation levels were assessed using MassCLEAVE™ technology (A) We measured the DNA methylation calls according to the MassCLEAVE™ technology, but using ASF (Supplementary Data). Known input ratios of completely methylated and unmethylated DNA fragments (referred to as 0, 5, 10, 25, 50, 75, 90 or 100% methylated input DNA; panels from upper left to lower right, respectively) were either analyzed immediately in a T7 transcription-RNase A digestion reaction, followed by a MALDI-TOF MS analysis (direct analysis, gray bars), or analyzed following an extra PCR step (PCR + analysis, black bars). For each panel, the methylation calls are arranged per fragment in an increasing mass order ( x -axis) and the measured methylation ratios ( y -axis) are given as means + SEM of five replicate measurements. The dotted vertical line indicates the 1700 Da threshold, and the horizontal dotted lines represent the input methylation levels. (B) Accuracy of DNA methylation calls by MassCLEAVE™ and PCR-induced bias. The left panel shows the mean DNA methylation calls of fragments with a mass over 1700 Da ( y -axis) plotted against the input levels ( x -axis), revealing a high correlation between input and output levels. The right panel shows the difference between the assay output and the known input (error) versus the input DNA methylation levels, revealing a small but consistent bias. The data for the direct analysis is plotted as a grey line and the data for PCR + analysis is shown as a dashed black line. Data are given as means + SEM of five replicate measurements. Similar accuracy and precision data for SCTR and INHBB CpG islands are given as Supplementary Data files.

    Article Snippet: A crucial step in the Sequenom® DNA methylation analysis is the PCR amplification, as a successful PCR is likely to result in a good MALDI-TOF-MS spectrum and thus in an accurate determination of the DNA methylation status of the fragments.

    Techniques: DNA Methylation Assay, Quantitation Assay, CpG Methylation Assay, Methylation, Mass Spectrometry, Polymerase Chain Reaction

    DNA methylation downstream of SALL4 TSS interferes with RNA polymerase II elongation ( a ) Diagram of SALL4 gene. TSS and exon1–4 are shown. ChIP assays with RNA polymerase II antibody, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0–D10, and SALL4 primers spanning different exons, as indicated. ( b ) RIP assays with RNA polymerase II antibody, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0 and D10, and SALL4 primers spanning exons 1 and 4, as indicated in (a).

    Journal: Oncogene

    Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

    doi: 10.1038/onc.2016.399

    Figure Lengend Snippet: DNA methylation downstream of SALL4 TSS interferes with RNA polymerase II elongation ( a ) Diagram of SALL4 gene. TSS and exon1–4 are shown. ChIP assays with RNA polymerase II antibody, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0–D10, and SALL4 primers spanning different exons, as indicated. ( b ) RIP assays with RNA polymerase II antibody, employing HepAD38 cells grown −/+ HBV replication by tetracycline removal for D0 and D10, and SALL4 primers spanning exons 1 and 4, as indicated in (a).

    Article Snippet: Since there is no report about DNA methylation regulating SALL4 expression in tumors, we retrieved array-based DNA methylation platform data (Illumina HumanMethylation450K BeadChip) from TCGA (The Cancer Genome Atlas, http://cancergenome.nih.gov/ ) (data not shown).

    Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation

    HBV infection induces DNA demethylation of SALL4 in HepG2 hNTCP cell line ( a ) Quantification of secreted HBeAg by ELISA (Left panel), and quantification of pregenomic RNA by QPCR (Right panel) after HBV infection for 6 days. ( b ) Quantification of SALL4 gene expression (SALL4A and SALL4B slicing variants) by QPCR after HBV infection for 4 and 6 days (D4, D6). ( c ) Bisulfite sequencing PCR results of SALL4 clones, using DNA from HepG2 hNTCP cells without (−) HBV infection (D0) or with (+) HBV infection at 4 and 6 days (D4 and D6). Open and closed circles denote unmethylated and methylated states, respectively. Error bars denote S.D. * P

    Journal: Oncogene

    Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

    doi: 10.1038/onc.2016.399

    Figure Lengend Snippet: HBV infection induces DNA demethylation of SALL4 in HepG2 hNTCP cell line ( a ) Quantification of secreted HBeAg by ELISA (Left panel), and quantification of pregenomic RNA by QPCR (Right panel) after HBV infection for 6 days. ( b ) Quantification of SALL4 gene expression (SALL4A and SALL4B slicing variants) by QPCR after HBV infection for 4 and 6 days (D4, D6). ( c ) Bisulfite sequencing PCR results of SALL4 clones, using DNA from HepG2 hNTCP cells without (−) HBV infection (D0) or with (+) HBV infection at 4 and 6 days (D4 and D6). Open and closed circles denote unmethylated and methylated states, respectively. Error bars denote S.D. * P

    Article Snippet: Since there is no report about DNA methylation regulating SALL4 expression in tumors, we retrieved array-based DNA methylation platform data (Illumina HumanMethylation450K BeadChip) from TCGA (The Cancer Genome Atlas, http://cancergenome.nih.gov/ ) (data not shown).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Methylation Sequencing, Polymerase Chain Reaction, Clone Assay, Methylation

    HBV replication induces DNA demethylation of SALL4 in HepAD38 cell line ( a ) PCR quantification of SALL4 mRNA in HepAD38 cells grown without (−) HBV replication (D0) or with (+) HBV replication by tetracycline removal for 5, 10, 20 days (D5–D20). Results are from three independent RNA isolations performed in identical triplicates. Error bars denote S.D. * P

    Journal: Oncogene

    Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

    doi: 10.1038/onc.2016.399

    Figure Lengend Snippet: HBV replication induces DNA demethylation of SALL4 in HepAD38 cell line ( a ) PCR quantification of SALL4 mRNA in HepAD38 cells grown without (−) HBV replication (D0) or with (+) HBV replication by tetracycline removal for 5, 10, 20 days (D5–D20). Results are from three independent RNA isolations performed in identical triplicates. Error bars denote S.D. * P

    Article Snippet: Since there is no report about DNA methylation regulating SALL4 expression in tumors, we retrieved array-based DNA methylation platform data (Illumina HumanMethylation450K BeadChip) from TCGA (The Cancer Genome Atlas, http://cancergenome.nih.gov/ ) (data not shown).

    Techniques: Polymerase Chain Reaction

    SALL4 re-expression in liver cancer cell lines and HCV-related HCCs via DNA demethylation ( a ) Methylation-specific PCR (MSP) assay for SALL4 CpG site 13 and CpG sites 24–26, employing genomic DNA from HepAD38 cells grown without (−) and with (+) HBV replication for 10 days, analyzed 2% agarose gel electrophoresis. Relative intensity quantified by ImageJ software is ratio of +/− HBV replication. ( b ) QRT-PCR of SALL4 mRNA expression employing RNA from HepAD38 cells grown in the absence of HBV replication, Huh7 and Hep3B cell lines. Data normalized to GAPDH. −ΔCt values are shown. ( c ) MSP assay for CpG site 13 and CpG24–26 using genomic DNA from HepAD38 (without HBV replication), Huh7 and Hep3B cells, analyzed by 2% agarose gel electrophoresis. Relative intensity quantified by ImageJ software is ratio of signal from Huh7 or Hep3B to HepAD38 cells. ( d ) and ( f ) QRT-PCR of SALL4 mRNA expression in HBV- and HCV- related liver tumors (T) vs. peritumoral tissue (PT); ( e ) and ( h ) MSP assay for methylation of CpG13 and CpG24–26 sites using genomic DNA of HBV- and HCV- related HCCs; relative intensity is ratio of T/PT. ( g ) Statistical analysis for methylation status of SALL4 CpG13 and CpG24–26 sites in patient samples with SALL4 High and SALL4 low mRNA expression, using unpaired t test; P

    Journal: Oncogene

    Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

    doi: 10.1038/onc.2016.399

    Figure Lengend Snippet: SALL4 re-expression in liver cancer cell lines and HCV-related HCCs via DNA demethylation ( a ) Methylation-specific PCR (MSP) assay for SALL4 CpG site 13 and CpG sites 24–26, employing genomic DNA from HepAD38 cells grown without (−) and with (+) HBV replication for 10 days, analyzed 2% agarose gel electrophoresis. Relative intensity quantified by ImageJ software is ratio of +/− HBV replication. ( b ) QRT-PCR of SALL4 mRNA expression employing RNA from HepAD38 cells grown in the absence of HBV replication, Huh7 and Hep3B cell lines. Data normalized to GAPDH. −ΔCt values are shown. ( c ) MSP assay for CpG site 13 and CpG24–26 using genomic DNA from HepAD38 (without HBV replication), Huh7 and Hep3B cells, analyzed by 2% agarose gel electrophoresis. Relative intensity quantified by ImageJ software is ratio of signal from Huh7 or Hep3B to HepAD38 cells. ( d ) and ( f ) QRT-PCR of SALL4 mRNA expression in HBV- and HCV- related liver tumors (T) vs. peritumoral tissue (PT); ( e ) and ( h ) MSP assay for methylation of CpG13 and CpG24–26 sites using genomic DNA of HBV- and HCV- related HCCs; relative intensity is ratio of T/PT. ( g ) Statistical analysis for methylation status of SALL4 CpG13 and CpG24–26 sites in patient samples with SALL4 High and SALL4 low mRNA expression, using unpaired t test; P

    Article Snippet: Since there is no report about DNA methylation regulating SALL4 expression in tumors, we retrieved array-based DNA methylation platform data (Illumina HumanMethylation450K BeadChip) from TCGA (The Cancer Genome Atlas, http://cancergenome.nih.gov/ ) (data not shown).

    Techniques: Expressing, Methylation, Polymerase Chain Reaction, MSP Assay, Agarose Gel Electrophoresis, Software, Quantitative RT-PCR

    SALL4 expression is associated with DNA demethylation in a subgroup of HBV-related HCCs ( a ) PCR quantification of SALL4 mRNA in HBV-related HCCs with mild (Left panel) and extreme increase (Right panel) of SALL4 expression. PT, peritumor; T, tumor. ( b ) Mapping of CpG dinucleotides in human SALL4 gene. The transcriptional start site (TSS), numbered CpG sites, OCT4 and STAT3 binding sites, and the position of bisulfite sequencing primers are indicated. ( c–e ) Bisulfite sequencing PCR of SALL4 clones, using DNA from patients #24 ( c ), #72 ( d ), and #75 ( e ), as indicated in Fig. 1a. Open and closed circles denote unmethylated and methylated states, respectively.

    Journal: Oncogene

    Article Title: DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection

    doi: 10.1038/onc.2016.399

    Figure Lengend Snippet: SALL4 expression is associated with DNA demethylation in a subgroup of HBV-related HCCs ( a ) PCR quantification of SALL4 mRNA in HBV-related HCCs with mild (Left panel) and extreme increase (Right panel) of SALL4 expression. PT, peritumor; T, tumor. ( b ) Mapping of CpG dinucleotides in human SALL4 gene. The transcriptional start site (TSS), numbered CpG sites, OCT4 and STAT3 binding sites, and the position of bisulfite sequencing primers are indicated. ( c–e ) Bisulfite sequencing PCR of SALL4 clones, using DNA from patients #24 ( c ), #72 ( d ), and #75 ( e ), as indicated in Fig. 1a. Open and closed circles denote unmethylated and methylated states, respectively.

    Article Snippet: Since there is no report about DNA methylation regulating SALL4 expression in tumors, we retrieved array-based DNA methylation platform data (Illumina HumanMethylation450K BeadChip) from TCGA (The Cancer Genome Atlas, http://cancergenome.nih.gov/ ) (data not shown).

    Techniques: Expressing, Polymerase Chain Reaction, Binding Assay, Methylation Sequencing, Clone Assay, Methylation

    DNA methylation at the three top significant CpGs in KDM5C mutations cases and population controls. DNA methylation microarray data at three CpG sites within CpG-rich promoters of three genes FBXL5 ( A ), SCMH1 ( B ) and CACYBP ( C ) as determined in 6 published studies using Illumina methylation27 array. AF (Aging in females, n = 93), AP1 (aging pediatric 1, n = 398), AP2 (aging pediatric 2, n = 79), CO (cancer ovarian, n = 257), DB (diabetes, n = 99), DS (Down syndrome, n = 21), K-C are controls from our study (N = 16), K-M are KDM5C mutations cases. For CO and DS only control samples were included.

    Journal: BMC Medical Genomics

    Article Title: Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

    doi: 10.1186/1755-8794-6-1

    Figure Lengend Snippet: DNA methylation at the three top significant CpGs in KDM5C mutations cases and population controls. DNA methylation microarray data at three CpG sites within CpG-rich promoters of three genes FBXL5 ( A ), SCMH1 ( B ) and CACYBP ( C ) as determined in 6 published studies using Illumina methylation27 array. AF (Aging in females, n = 93), AP1 (aging pediatric 1, n = 398), AP2 (aging pediatric 2, n = 79), CO (cancer ovarian, n = 257), DB (diabetes, n = 99), DS (Down syndrome, n = 21), K-C are controls from our study (N = 16), K-M are KDM5C mutations cases. For CO and DS only control samples were included.

    Article Snippet: B & C) Boxplots of Illumina DNA methylation data for two microrray probes within SCMH1 gene.

    Techniques: DNA Methylation Assay, Microarray

    DNA methylation at FBXL5, SCMH1 an d CACYBP promoters correlates with KDM5C/KDM5D dosage. Boxplots show DNA methylation levels for CpG sites within the FBXL5, SCMH1 and CACYBP promoters in blood in 7 group of samples with different dosage of KDM5C/KDM5D . CpG numbering corresponds to Figures 4 D, 6 D and 7 D. CACYBP CpG#2 boxplot is not shown, as no correlation with KDM5C/KDM5D dosage was found for this site. The Y axis is % of DNA methylation. The X axis shows groups of samples, numbered from 1 to 7. Information for each group regarding sex chromosome constitution, X-chromosome inactivation (XCI, Xa is active and Xi is inactive X-chromosome respectively), presence/absence of KDM5C mutation, functional KDM5C/KDM5D dosage, and number of samples is shown in the table beside the graph.

    Journal: BMC Medical Genomics

    Article Title: Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

    doi: 10.1186/1755-8794-6-1

    Figure Lengend Snippet: DNA methylation at FBXL5, SCMH1 an d CACYBP promoters correlates with KDM5C/KDM5D dosage. Boxplots show DNA methylation levels for CpG sites within the FBXL5, SCMH1 and CACYBP promoters in blood in 7 group of samples with different dosage of KDM5C/KDM5D . CpG numbering corresponds to Figures 4 D, 6 D and 7 D. CACYBP CpG#2 boxplot is not shown, as no correlation with KDM5C/KDM5D dosage was found for this site. The Y axis is % of DNA methylation. The X axis shows groups of samples, numbered from 1 to 7. Information for each group regarding sex chromosome constitution, X-chromosome inactivation (XCI, Xa is active and Xi is inactive X-chromosome respectively), presence/absence of KDM5C mutation, functional KDM5C/KDM5D dosage, and number of samples is shown in the table beside the graph.

    Article Snippet: B & C) Boxplots of Illumina DNA methylation data for two microrray probes within SCMH1 gene.

    Techniques: DNA Methylation Assay, Mutagenesis, Functional Assay

    Regional DNA methylation in FBXL5 promoter. A ) Screenshot from the UCSC genome browser showing location of FBXL5 promoter exon1, intron1, CpG island, Illumina microarray probes and pyrosequencing assays and tracks for H3K4me1 and H3K4me3 in lymphoblastoid cell line (GM12878) and ES cell line (H1 h-ES) (Broad Institute Histone data). B C ) Boxplots of Illumina DNA methylation data for two microrray probes within FBXL5 gene. The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively; the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. C are controls (N = 19), K are cases with KDM5C mutations (N = 10), V are cases with p.R1546Q sequence variant. D ) DNA methylation upstream of FBXL5 transcription start site as determined by pyrosequencing assays 1 2 (pyro1 2). The order of CpG sites are shown from the furthest upstream to the transcription start site. Each column is an average for each of three groups of 1)10 cases with KDM5C mutations (mutation), 2)19 controls (control), and 3) two individuals with the p.R1546Q variant of unknown significance (VUS). The arrow shows the CpG sites overlapping the microarray probe. P-values were determined by Kruskal-Wallis test between mutation cases and controls. **** is p

    Journal: BMC Medical Genomics

    Article Title: Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

    doi: 10.1186/1755-8794-6-1

    Figure Lengend Snippet: Regional DNA methylation in FBXL5 promoter. A ) Screenshot from the UCSC genome browser showing location of FBXL5 promoter exon1, intron1, CpG island, Illumina microarray probes and pyrosequencing assays and tracks for H3K4me1 and H3K4me3 in lymphoblastoid cell line (GM12878) and ES cell line (H1 h-ES) (Broad Institute Histone data). B C ) Boxplots of Illumina DNA methylation data for two microrray probes within FBXL5 gene. The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively; the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. C are controls (N = 19), K are cases with KDM5C mutations (N = 10), V are cases with p.R1546Q sequence variant. D ) DNA methylation upstream of FBXL5 transcription start site as determined by pyrosequencing assays 1 2 (pyro1 2). The order of CpG sites are shown from the furthest upstream to the transcription start site. Each column is an average for each of three groups of 1)10 cases with KDM5C mutations (mutation), 2)19 controls (control), and 3) two individuals with the p.R1546Q variant of unknown significance (VUS). The arrow shows the CpG sites overlapping the microarray probe. P-values were determined by Kruskal-Wallis test between mutation cases and controls. **** is p

    Article Snippet: B & C) Boxplots of Illumina DNA methylation data for two microrray probes within SCMH1 gene.

    Techniques: DNA Methylation Assay, Microarray, Sequencing, Variant Assay, Mutagenesis

    Sex-specific DNA methylation differences in FBXL5, CACYBP and SCMH1 promoters in blood and four brain regions. Boxplots show DNA methylation levels in three CpGs located in the promoters of FBXL5 , CACYBP and SCMH1 in 99 (48 males/51 females) blood samples (GSE20067), 133 (90 males/43 females) frontal cortex samples (FCTX), 127 (85 males/42 females) temporal cortex samples (TCTX), 121 (86 males/35 females) cerebellum samples (CBL) and 125 (87 males/38 females) pons samples from neurologically normal individuals (GSE15745). The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively, the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. P-values were calculated using Kruskal-Wallis test. **** is p

    Journal: BMC Medical Genomics

    Article Title: Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

    doi: 10.1186/1755-8794-6-1

    Figure Lengend Snippet: Sex-specific DNA methylation differences in FBXL5, CACYBP and SCMH1 promoters in blood and four brain regions. Boxplots show DNA methylation levels in three CpGs located in the promoters of FBXL5 , CACYBP and SCMH1 in 99 (48 males/51 females) blood samples (GSE20067), 133 (90 males/43 females) frontal cortex samples (FCTX), 127 (85 males/42 females) temporal cortex samples (TCTX), 121 (86 males/35 females) cerebellum samples (CBL) and 125 (87 males/38 females) pons samples from neurologically normal individuals (GSE15745). The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively, the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. P-values were calculated using Kruskal-Wallis test. **** is p

    Article Snippet: B & C) Boxplots of Illumina DNA methylation data for two microrray probes within SCMH1 gene.

    Techniques: DNA Methylation Assay

    Placental DNA methylation of the 11β-hydroxysteroid dehydrogenase type 2 ( HSD11B2 ) promoter. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Mean ± SD DNA methylation values are given for consecutive CpG sites in HSD11B2 assay 1 (A) and assay 2 (B) measured by bisulfite pyrosequencing.

    Journal: PLoS ONE

    Article Title: Early Onset Pre-Eclampsia Is Associated with Altered DNA Methylation of Cortisol-Signalling and Steroidogenic Genes in the Placenta

    doi: 10.1371/journal.pone.0062969

    Figure Lengend Snippet: Placental DNA methylation of the 11β-hydroxysteroid dehydrogenase type 2 ( HSD11B2 ) promoter. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Mean ± SD DNA methylation values are given for consecutive CpG sites in HSD11B2 assay 1 (A) and assay 2 (B) measured by bisulfite pyrosequencing.

    Article Snippet: Multiple genes associated with stress pathways and steroid production were associated with differentially methylated CpG sites in EOPET cases compared with controls based on the Illumina DNA methylation array ( ).

    Techniques: DNA Methylation Assay

    Placental DNA methylation of genes involved in steroidogenesis. DNA methylation at CpG sites within A) CYP11A1 , B) 3β-hydroxl-delta-5-steroid dehydrogenase type I ( HSD3B1 ), C) TEA domain family member 3 ( TEAD3 ) and D) CYP19 genes. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Median and interquartile ranges are given based on average assay CpG methylation measured by bisulfite pyrosequencing. *** P

    Journal: PLoS ONE

    Article Title: Early Onset Pre-Eclampsia Is Associated with Altered DNA Methylation of Cortisol-Signalling and Steroidogenic Genes in the Placenta

    doi: 10.1371/journal.pone.0062969

    Figure Lengend Snippet: Placental DNA methylation of genes involved in steroidogenesis. DNA methylation at CpG sites within A) CYP11A1 , B) 3β-hydroxl-delta-5-steroid dehydrogenase type I ( HSD3B1 ), C) TEA domain family member 3 ( TEAD3 ) and D) CYP19 genes. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Median and interquartile ranges are given based on average assay CpG methylation measured by bisulfite pyrosequencing. *** P

    Article Snippet: Multiple genes associated with stress pathways and steroid production were associated with differentially methylated CpG sites in EOPET cases compared with controls based on the Illumina DNA methylation array ( ).

    Techniques: DNA Methylation Assay, CpG Methylation Assay

    CpG sites on the Illumina Infinium HumanMethylation450 BeadChip array across the NR3C1 gene region. Upper graph: DNA methylation at each CpG site in control (n = 19) and early onset pre-eclampsia (EOPET; n = 19) placentae. Lower graph: enlargement of region associated with CpG island containing multiple alternative first exons (black boxes). The Illumina CpG probe identifier is indicated by cg# and the position of CpG sites on the graphs are not to scale. * P

    Journal: PLoS ONE

    Article Title: Early Onset Pre-Eclampsia Is Associated with Altered DNA Methylation of Cortisol-Signalling and Steroidogenic Genes in the Placenta

    doi: 10.1371/journal.pone.0062969

    Figure Lengend Snippet: CpG sites on the Illumina Infinium HumanMethylation450 BeadChip array across the NR3C1 gene region. Upper graph: DNA methylation at each CpG site in control (n = 19) and early onset pre-eclampsia (EOPET; n = 19) placentae. Lower graph: enlargement of region associated with CpG island containing multiple alternative first exons (black boxes). The Illumina CpG probe identifier is indicated by cg# and the position of CpG sites on the graphs are not to scale. * P

    Article Snippet: Multiple genes associated with stress pathways and steroid production were associated with differentially methylated CpG sites in EOPET cases compared with controls based on the Illumina DNA methylation array ( ).

    Techniques: DNA Methylation Assay

    Model of altered stress and hormonal signalling gene-pathways in pre-eclampsia. Left hand side: summary of altered DNA methylation at specific CpG sites across candidate genes, where black and white circles indicate gain or loss of methylation, respectively. Coding regions are indicated by black rectangles and transcription factor (TF) binding motifs are represented by grey circles and include yin yang 1 (YY1; putative), activated protein 2 (AP2; known) and transcription enhancer factor 5 (TEF5; known). Bent lines represent transcription start sites: arrow or blunt heads hypothesise increased or decreased expression, respectively. Green bars represent CpG islands. Annotations are of approximate location and distance. Right hand side: hypothesised model of the downstream effects of altered placental DNA methylation at candidate genes. NR3C1 : nuclear receptor subfamily 3, group C, member 1, CRH : corticotropin releasing hormone, CRHBP : CRH binding protein, HSD3B1 ∶3β-hydroxy-delta-5-steroid dehydrogenase type 1, TEAD3 : TEA domain family member 3, GR: glucocorticoid receptor, HSD11B2 ∶11β-hydroxysteroid dehydrogenase type 2, P450scc: P450 side chain cleavage, 3βHSD1∶3β-hydroxysteroid dehydrogenase type 1.

    Journal: PLoS ONE

    Article Title: Early Onset Pre-Eclampsia Is Associated with Altered DNA Methylation of Cortisol-Signalling and Steroidogenic Genes in the Placenta

    doi: 10.1371/journal.pone.0062969

    Figure Lengend Snippet: Model of altered stress and hormonal signalling gene-pathways in pre-eclampsia. Left hand side: summary of altered DNA methylation at specific CpG sites across candidate genes, where black and white circles indicate gain or loss of methylation, respectively. Coding regions are indicated by black rectangles and transcription factor (TF) binding motifs are represented by grey circles and include yin yang 1 (YY1; putative), activated protein 2 (AP2; known) and transcription enhancer factor 5 (TEF5; known). Bent lines represent transcription start sites: arrow or blunt heads hypothesise increased or decreased expression, respectively. Green bars represent CpG islands. Annotations are of approximate location and distance. Right hand side: hypothesised model of the downstream effects of altered placental DNA methylation at candidate genes. NR3C1 : nuclear receptor subfamily 3, group C, member 1, CRH : corticotropin releasing hormone, CRHBP : CRH binding protein, HSD3B1 ∶3β-hydroxy-delta-5-steroid dehydrogenase type 1, TEAD3 : TEA domain family member 3, GR: glucocorticoid receptor, HSD11B2 ∶11β-hydroxysteroid dehydrogenase type 2, P450scc: P450 side chain cleavage, 3βHSD1∶3β-hydroxysteroid dehydrogenase type 1.

    Article Snippet: Multiple genes associated with stress pathways and steroid production were associated with differentially methylated CpG sites in EOPET cases compared with controls based on the Illumina DNA methylation array ( ).

    Techniques: DNA Methylation Assay, Methylation, Binding Assay, Expressing

    Placental DNA methylation of genes involved in cortisol signalling and bioavailability. DNA methylation at CpG sites within A) exon 1D promoter of the nuclear receptor subfamily 3, group C, member 1 ( NR3C1 ), B) corticotropin releasing hormone ( CRH ) and C) CRH binding protein ( CRHBP ) genes. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Median and interquartile ranges are given based on average assay CpG methylation measured by bisulfite pyrosequencing. * P

    Journal: PLoS ONE

    Article Title: Early Onset Pre-Eclampsia Is Associated with Altered DNA Methylation of Cortisol-Signalling and Steroidogenic Genes in the Placenta

    doi: 10.1371/journal.pone.0062969

    Figure Lengend Snippet: Placental DNA methylation of genes involved in cortisol signalling and bioavailability. DNA methylation at CpG sites within A) exon 1D promoter of the nuclear receptor subfamily 3, group C, member 1 ( NR3C1 ), B) corticotropin releasing hormone ( CRH ) and C) CRH binding protein ( CRHBP ) genes. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Median and interquartile ranges are given based on average assay CpG methylation measured by bisulfite pyrosequencing. * P

    Article Snippet: Multiple genes associated with stress pathways and steroid production were associated with differentially methylated CpG sites in EOPET cases compared with controls based on the Illumina DNA methylation array ( ).

    Techniques: DNA Methylation Assay, Binding Assay, CpG Methylation Assay