Journal: Clinical Epigenetics

Article Title: Promoter methylation changes in ALOX12 and AIRE1 : novel epigenetic markers for atherosclerosis

doi: 10.1186/s13148-020-00846-0

Figure Lengend Snippet: Promoter CpG islands and regions for the pyrosequencing of the five target genes identified after methylation-specific PCR ( a ) and methylation levels of the genes in plaques (P) and non-plaque (NP) intima of common carotid arteries in 20 cadavers ( b ). Correlation between promoter methylation and expression of AIRE1 ( c ) and ALOX12 ( d ) in 18 carotid endarterectomy plaques. Open bar, exon 1 region; closed bar, the regions targeted for bisulfite pyrosequencing; open bar in the middle of closed bar; sequencing region, arrow; transcriptional start site of each gene, * p < 0.05 on paired t test

Article Snippet: We used bisulfite pyrosequencing primer sets for AIRE1 (AIRE1-F; biotin-5′-GGGTAGTTTTTGTTAGGGTTTTGA-3′ and AIRE1-R; 5′-TCACCCACCAAAACAACTACCTTA-3′ for PCR and AIRE1-S; 5′-TCAAAAAACTCTCACTTCC-3′ for bisulfite pyrosequencing) and NEOT1 (NEOT1-F; biotin-5′-GTGGGGGGTTTAATTGTAAGAAGT-3′ and NETO1-R; 5′-TCTCTACCTCCCACCCCTTCTTT-3′ for PCR and NETO1-S; 5′-CCCCTTCTTTTCCAT-3′ for bisulfite pyrosequencing) (Fig. a).

Techniques: Methylation, Expressing, Sequencing

Journal: Clinical Epigenetics

Article Title: Promoter methylation changes in ALOX12 and AIRE1 : novel epigenetic markers for atherosclerosis

doi: 10.1186/s13148-020-00846-0

Figure Lengend Snippet: Immunofluorescence staining for AIRE1 and infiltrated CD4(+)-T cells and CD14(+)-monocytes in atherosclerotic plaque and non-plaque intima of common carotid artery of a 54-year-old man. White arrow, cells co-stained with CD14 and AIRE1 antibodies

Article Snippet: We used bisulfite pyrosequencing primer sets for AIRE1 (AIRE1-F; biotin-5′-GGGTAGTTTTTGTTAGGGTTTTGA-3′ and AIRE1-R; 5′-TCACCCACCAAAACAACTACCTTA-3′ for PCR and AIRE1-S; 5′-TCAAAAAACTCTCACTTCC-3′ for bisulfite pyrosequencing) and NEOT1 (NEOT1-F; biotin-5′-GTGGGGGGTTTAATTGTAAGAAGT-3′ and NETO1-R; 5′-TCTCTACCTCCCACCCCTTCTTT-3′ for PCR and NETO1-S; 5′-CCCCTTCTTTTCCAT-3′ for bisulfite pyrosequencing) (Fig. a).

Techniques: Immunofluorescence, Staining

Journal: Clinical Epigenetics

Article Title: Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction

doi: 10.1186/s13148-019-0630-4

Figure Lengend Snippet: Analysis of imprinted methylation at ubiquitous DMRs using HM450k methylation arrays. a The left side is a heatmap of absolute methylation ( β values) for individual Infinium probes mapping to 35 DMRs in 67 placenta samples. Samples with abnormal methylation across a DMR are highlighted by yellow boxes. The right side of the figure reveals methylation difference according to severity, with blue and red representing hypo- and hypermethylation, respectively. Samples are classified by both the presence of pre-eclampsia (black yes; gray no) and fetal growth parameters (appropriate for gestational age light gray; SGA dark gray; IUGR black). *Adjacent to the DMR name indicates somatic establishment of methylation. b Pyrosequencing confirmation of the aberrant methylation profiles identified using HM450k arrays, as well as the quantification of additional placenta samples. The violin plots used include the median (white dot) and the interquartile range (black rectangle), with hypomethylated samples identified using the HM450k array platform highlighted as green data points, while hypermethylated samples are in red. The non-parametric Mann-Whitney-Wilcoxon test was used to calculate the statistical significance of the differences between IUGR and control groups (ns indicated no significance, p > 0.05). c Pyrosequencing quantification in cord blood samples. The violin plots show the distribution of methylation for 16 controls. Samples with aberrant placenta methylation profiles are highlighted. d Quantitative RT-PCR for transcripts regulated by affected DMRs. The violin plots represent the expression levels of 50 control placenta samples with normal birthweight and methylation. Samples with hypomethylation are highlighted as green data points, while hypermethylated samples are in red. e Allelic expression analysis was performed for the H19 transcript using the rs2839704 SNP, with allelic contributions quantified by pyrosequencing

Article Snippet: Pyrosequencing: Standard bisulphite PCR was used to determine the methylation in a confirmatory cohort (see Additional file for details).

Techniques: Methylation, MANN-WHITNEY, Quantitative RT-PCR, Expressing

Journal: Clinical Epigenetics

Article Title: Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction

doi: 10.1186/s13148-019-0630-4

Figure Lengend Snippet: Detailed characterization of histone modifications within placenta-specific DMRs in samples with polymorphic imprinting. Schematic representation of a the LIN28B loci, indicating the position of transcripts and CpG islands incorporating the DMRs. The methylation of the two placenta samples analyzed was assessed by bisulphite PCR and sub-cloning. Each circle represents a single CpG on a DNA strand. (•) methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence with the genotype indicated for the heterozygous SNP incorporated into the amplicon. b Quantitative PCR targeting the LIN28B DMR in ChIP material. Precipitations were normalised to GAPDH promoter (H3K4me2/3) and SAT2 repeats (H3K9me2/3). The graphs represent the mean values (± standard deviation). For each placenta sample, values are the mean of at least three independent ChIP experiments, each in triplicate. c The allelic distribution of each mark was determined by direct sequencing of the PCR product encompassing the heterozygous SNP used for the methylation analysis. The percentage of allelic enrichment is shown under the electropherograms. d Diagram of the R3HCC1 loci and the methylation bisulphite PCR profiles of the three analyzed samples. e , f The quantitative and allelic ChIP results for the R3HCC1 DMR, respectively, which were analyzed in the same way as for LIN28B

Article Snippet: Pyrosequencing: Standard bisulphite PCR was used to determine the methylation in a confirmatory cohort (see Additional file for details).

Techniques: Methylation, Subcloning, Clone Assay, Sequencing, Amplification, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Clinical Epigenetics

Article Title: Differences in expression rather than methylation at placenta-specific imprinted loci is associated with intrauterine growth restriction

doi: 10.1186/s13148-019-0630-4

Figure Lengend Snippet: Epigenetic and transcriptional description of the GPR1-AS1-ADAM23 locus in IUGR placentas. a Schematic representation of the GPR1 - AS1-ADAM23 imprinted locus on chromosome 2, indicating the position of the transcripts and CpG islands incorporating the DMRs. b Characterization of the DMRs and promoter CpG islands in placenta biopsies. Each circle represents a single CpG on a DNA strand. (•) methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence with the genotype indicated for heterozygous SNP incorporated into the amplicon. c Quantitative PCR on ChIP material. Precipitations were normalized to GAPDH promoter (H3K4me2/3) and SAT2 repeats (H3K9me2/3). The graphs represent the mean values (± standard deviation). For each placenta sample, values are the mean of at least three independent ChIP experiments, each in triplicate. d The allelic distribution of each mark was determined by direct sequencing of the PCR product encompassing heterozygous SNPs. e Quantification of expression levels of GPR1-AS1 , ZDBF2 , and ADAM23 using microfluidic-based RT-qPCR. The results are presented as violin plots, with the median (white dot), mean (red line), and the interquartile range (black rectangle) shown. All expression levels were normalised to the mean of the RPL19 housekeeping gene. To determine the statistical significance of the difference between the IUGR and control groups, Student’s two-tailed t test was used and p values are indicated over the horizontal comparison lines. f Pyrosequencing quantification of the GPR1 -AS1 and ZDBF2 DMRs. The violin plots include the median (white dot) mean (red line) and the interquartile range (black rectangle). The non-parametric Mann-Whitney-Wilcoxon test was used to calculate the statistical significance of the differences between IUGR and control groups (ns indicated no significance, p > 0.05)

Article Snippet: Pyrosequencing: Standard bisulphite PCR was used to determine the methylation in a confirmatory cohort (see Additional file for details).

Techniques: Methylation, Clone Assay, Sequencing, Amplification, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Targeting the Immune system and Epigenetic Landscape of Urological Tumors

doi: 10.3390/ijms21030829

Figure Lengend Snippet: Immunoepigenetic-based studies in bladder cancer.

Article Snippet: Methylation , DEFB1 (mediator of innate immunity) , Epigenetic regulation of DEFB1 by promoter methylation , Bisulfite-sequencing Pyrosequencing RT-qPCR IHC WB , Tissues ( n = 60 patients) + in vitro (cell lines) , Lee J, 2016 [ ] .

Techniques: Methylation, Expressing, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, In Vitro, CpG Methylation Assay, Knock-Out, CRISPR, In Vivo, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Targeting the Immune system and Epigenetic Landscape of Urological Tumors

doi: 10.3390/ijms21030829

Figure Lengend Snippet: Immunoepigenetic-based studies in Kidney Cancer.

Article Snippet: Methylation , DEFB1 (mediator of innate immunity) , Epigenetic regulation of DEFB1 by promoter methylation , Bisulfite-sequencing Pyrosequencing RT-qPCR IHC WB , Tissues ( n = 60 patients) + in vitro (cell lines) , Lee J, 2016 [ ] .

Techniques: Methylation, Activity Assay, Microarray, In Vitro, In Vivo, Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Targeting the Immune system and Epigenetic Landscape of Urological Tumors

doi: 10.3390/ijms21030829

Figure Lengend Snippet: Immunoepigenetic-based studies in Prostate Cancer.

Article Snippet: Methylation , DEFB1 (mediator of innate immunity) , Epigenetic regulation of DEFB1 by promoter methylation , Bisulfite-sequencing Pyrosequencing RT-qPCR IHC WB , Tissues ( n = 60 patients) + in vitro (cell lines) , Lee J, 2016 [ ] .

Techniques: Methylation, Expressing, Northern Blot, In Vitro, Affinity Chromatography, Methylation Sequencing, Migration, In Vivo, Lysis, Blocking Assay, Infection, DNA Sequencing, Ex Vivo, Microarray, Enzyme-linked Immunosorbent Assay, Inhibition, Immunohistochemistry-IF