Journal: Alcoholism, clinical and experimental research

Article Title: Hypermethylation of proopiomelanocortin and period 2 genes in blood are associated with greater subjective and behavioral motivation for alcohol in humans

doi: 10.1111/acer.13932

Figure Lengend Snippet: PER2 (A) and POMC gene expression (B) and PER2 (C) and POMC gene methylation (D, E) levels in each of the three drinking groups are shown. Gene expression levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Gene methylation levels were measured by methylation specific PCR (MSP) and was represented as relative DNA methylation (C & D). POMC DNA methylation was additionally verified by pyrosequencing of the methylated DNA within the promoter area of the gene (Fig. E), Pyrosequencing assay for PER2 was not successful possibly because of high density of CpG residue. Data are represented as Mean ± SEM. Number of samples in each group is shown between brackets under the group heading on the X axis or within the figures. Statistically significant differences between groups are shown by lines with p values on the top of bar graphs.

Article Snippet: Human highly methylated and low methylated control DNA (Epigen DX, Worcester MA) were subjected to bisulfite conversion and were used for preparing the standard curve. qRT-PCR was performed using SYBR green master mix with specific primers and bisulfite converted DNA as template. qRT-PCR was performed using a program at 95°C for 5 min followed by 50 cycles of 95°C for 30sec, 60°C for 1min, 72°C for 1 min.

Techniques: Gene Expression, Methylation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, DNA Methylation Assay, Pyrosequencing Assay, Residue

Journal: The Journal of Biological Chemistry

Article Title: Epithelial Cell Adhesion Molecule Regulation Is Associated with the Maintenance of the Undifferentiated Phenotype of Human Embryonic Stem Cells *

doi: 10.1074/jbc.M109.077081

Figure Lengend Snippet: Methylation status of EpCAM promoter regions in undifferentiated and differentiated hESCs. A, schematic representation of the EpCAM gene promoter region. Primers for MSP and bisulfite sequencing used in the study are indicated. B, MSP analysis of the EpCAM gene promoter region in undifferentiated and differentiated H9 cells. The PCR products that were methylated (M) were generated by methylation-specific primers, and those that were unmethylated (U) were generated by primers specific for unmethylated DNA. C, mapping the methylation status of the CpG islands in the promoter region of the EpCAM gene by bisulfite sequencing. Each row of squares represents a single plasmid cloned and sequenced from PCR products generated from amplification of bisulfite-treated DNA. Open squares, unmethylated cytosines; filled squares, methylated cytosines. Most CpGs in the promoter region in both undifferentiated and differentiated H9 cells were unmethylated.

Article Snippet: Completely methylated and unmethylated control genomic DNA was purchased from Qiagen (Qiagen Inc., Valencia, CA).

Techniques: Methylation, Methylation Sequencing, Generated, Plasmid Preparation, Clone Assay, Amplification