genomic dna - human adult normal tissue: peripheral blood leukocyte #cp-d300d1234148  (BioChain Institute)

Journal: Clinical Epigenetics

Article Title: Platelet mitochondrial DNA methylation: a potential new marker of cardiovascular disease

doi: 10.1186/s13148-015-0078-0

Figure Lengend Snippet: A representative gel following PCR amplification of mitochondrial and nuclear DNA sequences. Amplification of MT-CO1 , CDH1 , CDKN2A , and LINE-1 in extracted mtDNA (lanes 1 to 4) and control DNA from human adult normal peripheral blood leukocyte (Biochain, Newark, CA, USA) (lanes 6 to 9) were visualized on a 2% agarose gel. The fifth lane contains a DNA size marker.

Article Snippet: Amplification of MT-CO1 , CDH1 , CDKN2A , and LINE-1 in extracted mtDNA (lanes 1 to 4) and control DNA from human adult normal peripheral blood leukocyte (Biochain, Newark, CA, USA) (lanes 6 to 9) were visualized on a 2% agarose gel.

Techniques: Amplification, Control, Agarose Gel Electrophoresis, Marker

Journal: Clinical Epigenetics

Article Title: Platelet mitochondrial DNA methylation: a potential new marker of cardiovascular disease

doi: 10.1186/s13148-015-0078-0

Figure Lengend Snippet: Technical replicates and quantitative validation of mtDNA pyrosequencing assays. Spearman correlation coefficients ( r ) of the technical replicates (replicates 1 and 2) for the designed pyrosequencing assays were (a) 0.97 for MT-CO1 , (b) 0.93 for MT-CO2 , and (c) 0.76 for MT-CO3 . The plot curve is displayed with a 95% confidence band. The quantitative validation of 0% and 100% methylated DNA for the (d) MT-CO1 , (e) MT-CO2 , and (f) MT-CO3 assays (red circle) were compared with 0% and 100% control oligonucleotides (black rectangle).

Article Snippet: Amplification of MT-CO1 , CDH1 , CDKN2A , and LINE-1 in extracted mtDNA (lanes 1 to 4) and control DNA from human adult normal peripheral blood leukocyte (Biochain, Newark, CA, USA) (lanes 6 to 9) were visualized on a 2% agarose gel.

Techniques: Biomarker Discovery, Methylation, Control

Journal: Clinical Epigenetics

Article Title: Platelet mitochondrial DNA methylation: a potential new marker of cardiovascular disease

doi: 10.1186/s13148-015-0078-0

Figure Lengend Snippet: A representative gel following PCR amplification of mitochondrial and nuclear DNA sequences. Amplification of MT-CO1 , CDH1 , CDKN2A , and LINE-1 in extracted mtDNA (lanes 1 to 4) and control DNA from human adult normal peripheral blood leukocyte (Biochain, Newark, CA, USA) (lanes 6 to 9) were visualized on a 2% agarose gel. The fifth lane contains a DNA size marker.

Article Snippet: In addition to extracted mtDNA, human adult normal peripheral blood leukocyte DNA (Biochain, Newark, CA, USA) was used as a control.

Techniques: Amplification, Agarose Gel Electrophoresis, Marker

Journal: Clinical Epigenetics

Article Title: Platelet mitochondrial DNA methylation: a potential new marker of cardiovascular disease

doi: 10.1186/s13148-015-0078-0

Figure Lengend Snippet: Technical replicates and quantitative validation of mtDNA pyrosequencing assays. Spearman correlation coefficients ( r ) of the technical replicates (replicates 1 and 2) for the designed pyrosequencing assays were (a) 0.97 for MT-CO1 , (b) 0.93 for MT-CO2 , and (c) 0.76 for MT-CO3 . The plot curve is displayed with a 95% confidence band. The quantitative validation of 0% and 100% methylated DNA for the (d) MT-CO1 , (e) MT-CO2 , and (f) MT-CO3 assays (red circle) were compared with 0% and 100% control oligonucleotides (black rectangle).

Article Snippet: In addition to extracted mtDNA, human adult normal peripheral blood leukocyte DNA (Biochain, Newark, CA, USA) was used as a control.

Techniques: Methylation

Journal: Nucleic Acids Research

Article Title: Laser capture microdissection–reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse

doi: 10.1093/nar/gkt230

Figure Lengend Snippet: LCM-RRBS workflow. A complex tissue is dissected using LCM. Extracted DNA is digested by the methylation-insensitive enzyme MspI, end repaired and ligated with methylated Illumina adapters. After bisulfite conversion, each sample is ‘barcoded’ by introducing a sample-specific index (shown as green, blue or violet boxes) through low-cycle PCR. Samples are pooled and loaded onto a high-percentage gel for fragment separation and size selection. Using universal primers, the final library is amplified and sequenced on the Illumina platform.

Article Snippet: RRBS was performed on 400 ng of commercially purchased leukocyte genomic DNA (D1234148, Amsbio) and genomic DNA extracted from a mouse liver as previously described ( ).

Techniques: Methylation, Selection, Amplification

Journal: Nucleic Acids Research

Article Title: Laser capture microdissection–reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse

doi: 10.1093/nar/gkt230

Figure Lengend Snippet: LCM-RRBS is reproducible and robust across 1 ng extracted from bulk fresh frozen and FFPE samples. CGI methylation (top panels) and the methylation at 2-kb regions flanking the TSS (bottom panels) were compared between ( A ) 1 ng (LCM-RRBS) and 400 ng of purified leukocyte genomic DNA (RRBS), and ( B ) 1 ng of FFPE DNA and 1 ng of fresh frozen genomic DNA extracted from the same endometrial tumor (LCM-RRBS).

Article Snippet: RRBS was performed on 400 ng of commercially purchased leukocyte genomic DNA (D1234148, Amsbio) and genomic DNA extracted from a mouse liver as previously described ( ).

Techniques: Methylation, Purification

Journal: Nucleic Acids Research

Article Title: Laser capture microdissection–reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse

doi: 10.1093/nar/gkt230

Figure Lengend Snippet: LCM-RRBS is robust across microdissected samples collected from fresh frozen and FFPE tissues. Fresh frozen and FFPE mouse liver was collected for DNA methylation profiling. LCM was used to collect tissue from areas ranging in size from 20 to 2 mm 2 . CGI methylation (top panels) and methylation at 2-kb regions flanking the TSS (bottom panels) were compared between ( A ) fresh frozen samples (LCM-RRBS) and 400 ng of purified mouse liver genomic DNA (RRBS), and ( B ) FFPE samples (LCM-RRBS) and 400 ng of purified mouse liver genomic DNA (RRBS).

Article Snippet: RRBS was performed on 400 ng of commercially purchased leukocyte genomic DNA (D1234148, Amsbio) and genomic DNA extracted from a mouse liver as previously described ( ).

Techniques: DNA Methylation Assay, Methylation, Purification

Journal: Nucleic Acids Research

Article Title: Laser capture microdissection–reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse

doi: 10.1093/nar/gkt230

Figure Lengend Snippet: DNA methylation profiling of GDX-induced adrenocortical neoplasms and adjacent normal tissue using LCM-RRBS. The adrenal glands of three ovariectomized DBA/2J mice were fresh frozen in Tissue-tek O.C.T. compound, cryosectioned and stained. Shown are representative cryosections pre- and post-LCM. Normal cells in the zona fasciculata contain large lipid droplets that are easily recognized. In contrast, neoplastic cells distort the normal adrenal zonal architecture and contain relatively few lipid droplets. The microdissected normal tissue included zona glomerulosa and zona fasciculata cells; care was taken to avoid dissection of X-zone (X), medulla (M) or capsule cells, as these cell types have distinct developmental origins , and therefore may have different epigenetic fingerprints. An average of 5.5 mm 2 of neoplastic (red) and normal (green) tissue per adrenal pair was collected and analyzed using LCM-RRBS.

Article Snippet: RRBS was performed on 400 ng of commercially purchased leukocyte genomic DNA (D1234148, Amsbio) and genomic DNA extracted from a mouse liver as previously described ( ).

Techniques: DNA Methylation Assay, Staining, Dissection

Journal: Nucleic Acids Research

Article Title: Laser capture microdissection–reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse

doi: 10.1093/nar/gkt230

Figure Lengend Snippet: Validation of differentially methylated promoters. The DNA methylation of one hypermethylated and three hypomethylated promoters was interrogated by BSP and sequencing across enriched neoplastic and normal samples. All genes show a statistically significant difference (Fisher’s exact test, P < 10 −15 ) in DNA methylation using BSP. Each colored box represents an individual CpG dinucleotide within a 2-kb region centered around the TSS. High (yellow), moderate (black), low (blue) and undetermined methylation levels are shown for each CpG. The mean methylation of each region interrogated is shown to the right of each heatmap. The red box indicates the region of the promoter that was interrogated by LCM-RRBS and BSP.

Article Snippet: RRBS was performed on 400 ng of commercially purchased leukocyte genomic DNA (D1234148, Amsbio) and genomic DNA extracted from a mouse liver as previously described ( ).

Techniques: Methylation, DNA Methylation Assay, Sequencing