quantitative bisulfite pyrosequencing analysis of promoter dna methylation  (Pyrosequencing Inc)

Journal:

Article Title: DNA hypermethylation regulates the expression of members of the Mu-class Glutathione-S-Transferases and Glutathione Peroxidases in Barrett's adenocarcinoma

doi: 10.1136/gut.2007.146290

Figure Lengend Snippet: mRNA expression and DNA methylation of GSTs and GPXs in Barrett's adenocarcinomas

Article Snippet: The remaining 14 genes with CpG islands were analyzed by qRT-PCR and quantitative bisulfite pyrosequencing analysis of promoter DNA methylation. table ft1 table-wrap mode="anchored" t5 caption a7 Class Gene Cytoband Promoter CpG island * Representative mRNA ACC Representative protein ACC Subcellular location GSTs Alpha (a) GSTA1 6p12 no NM_145740 NP_665683 cytoplasm GSTA2 6p12.1 no NM_000846 NP_000837 cytoplasm GSTA3 6p12 no NM_000847 NP_000838 cytoplasm GSTA4 6p12 yes NM_001512 NP_001503 cytoplasm GSTA5 6p12.1 no NM_153699 NP_714543 cytoplasm (by similarity).

Techniques: Expressing, DNA Methylation Assay

Journal:

Article Title: DNA hypermethylation regulates the expression of members of the Mu-class Glutathione-S-Transferases and Glutathione Peroxidases in Barrett's adenocarcinoma

doi: 10.1136/gut.2007.146290

Figure Lengend Snippet: A) DNA methylation profiles of 20 normal esophagus mucosae (NS), 17 normal gastric mucosae (NG), 11 Barrett's esophagus (BE) and 11 Barrett's dysplasia (BD). B) DNA methylation profiles of 100 Barrett's adenocarcinomas (BACs). The DNA methylation was quantified using pyrosequencing technology. The data are presented as an average methylation percentage of the analyzed CpG nucleotides for each gene. The details of the primers and the relationship between the transcription start site and the regions that were analyzed are given in supplementary Table 2 and supplementary Figure 1. The grid and the color codes are given at the bottom of the figure. C) A summary graph showing the frequencies of DNA methylation. GSTM5 is not shown since it showed more than 10% methylation in all normal and tumor samples.

Article Snippet: The remaining 14 genes with CpG islands were analyzed by qRT-PCR and quantitative bisulfite pyrosequencing analysis of promoter DNA methylation. table ft1 table-wrap mode="anchored" t5 caption a7 Class Gene Cytoband Promoter CpG island * Representative mRNA ACC Representative protein ACC Subcellular location GSTs Alpha (a) GSTA1 6p12 no NM_145740 NP_665683 cytoplasm GSTA2 6p12.1 no NM_000846 NP_000837 cytoplasm GSTA3 6p12 no NM_000847 NP_000838 cytoplasm GSTA4 6p12 yes NM_001512 NP_001503 cytoplasm GSTA5 6p12.1 no NM_153699 NP_714543 cytoplasm (by similarity).

Techniques: DNA Methylation Assay, Methylation

Journal:

Article Title: DNA hypermethylation regulates the expression of members of the Mu-class Glutathione-S-Transferases and Glutathione Peroxidases in Barrett's adenocarcinoma

doi: 10.1136/gut.2007.146290

Figure Lengend Snippet: The percentages of DNA methylation of the GPX3, GPX7, GSTM2, GSTM3, and GSTM5 genes were determined by quantitative bisulfite pyrosequencing. The horizontal bars locate the median of DNA methylation level. The DNA methylation levels were analyzed using the Wilcoxon Rank Sum test to determine the statistical significance. The tumor and premalignant (BE and BD) samples were compared to normal samples (NS + NG). A P value of ≤.05 was considered statistically significant.

Article Snippet: The remaining 14 genes with CpG islands were analyzed by qRT-PCR and quantitative bisulfite pyrosequencing analysis of promoter DNA methylation. table ft1 table-wrap mode="anchored" t5 caption a7 Class Gene Cytoband Promoter CpG island * Representative mRNA ACC Representative protein ACC Subcellular location GSTs Alpha (a) GSTA1 6p12 no NM_145740 NP_665683 cytoplasm GSTA2 6p12.1 no NM_000846 NP_000837 cytoplasm GSTA3 6p12 no NM_000847 NP_000838 cytoplasm GSTA4 6p12 yes NM_001512 NP_001503 cytoplasm GSTA5 6p12.1 no NM_153699 NP_714543 cytoplasm (by similarity).

Techniques: DNA Methylation Assay

Journal:

Article Title: DNA hypermethylation regulates the expression of members of the Mu-class Glutathione-S-Transferases and Glutathione Peroxidases in Barrett's adenocarcinoma

doi: 10.1136/gut.2007.146290

Figure Lengend Snippet: This figure demonstrates the methylation levels of individual CpG nucleotides in a representative set of matched tumor, normal, BE, and BD samples from the same patients. Each circle represents one CpG site in the promoter region. The number on the left side represents the samples’ code, as shown in Figure 1. The GSTM3 genes is not shown since only a few samples showed DNA methylation, see Figure 1.

Article Snippet: The remaining 14 genes with CpG islands were analyzed by qRT-PCR and quantitative bisulfite pyrosequencing analysis of promoter DNA methylation. table ft1 table-wrap mode="anchored" t5 caption a7 Class Gene Cytoband Promoter CpG island * Representative mRNA ACC Representative protein ACC Subcellular location GSTs Alpha (a) GSTA1 6p12 no NM_145740 NP_665683 cytoplasm GSTA2 6p12.1 no NM_000846 NP_000837 cytoplasm GSTA3 6p12 no NM_000847 NP_000838 cytoplasm GSTA4 6p12 yes NM_001512 NP_001503 cytoplasm GSTA5 6p12.1 no NM_153699 NP_714543 cytoplasm (by similarity).

Techniques: Methylation, DNA Methylation Assay

Journal:

Article Title: DNA hypermethylation regulates the expression of members of the Mu-class Glutathione-S-Transferases and Glutathione Peroxidases in Barrett's adenocarcinoma

doi: 10.1136/gut.2007.146290

Figure Lengend Snippet: A) mRNA expression of the GPX3, GPX7, GSTM2, GSTM3, and GSTM5 genes in normal (n=30) and primary Barrett's adenocarcinoma (n=66). The mRNA expression fold was determined by real-time RT-PCR and normalized to the average value of all the normal samples as described in materials and methods. Each open and black diamond represents a normal and tumor sample respectively. B) The mRNA expression fold is shown for methylated (black triangle) and unmethylated (open triangle) tumors. The horizontal bars in A and B locate the median expression fold. The statistical significance was determined by Wilcoxon Rank Sum test. C) The Spearman correlation analysis between DNA methylation level and mRNA expression fold in all 96 samples in A is depicted for each gene. Significant correlations were found for GPX3 (P<.0001), GPX7 (P=.002), GSTM2 (P=.0001), and GSTM5 (P=.01).

Article Snippet: The remaining 14 genes with CpG islands were analyzed by qRT-PCR and quantitative bisulfite pyrosequencing analysis of promoter DNA methylation. table ft1 table-wrap mode="anchored" t5 caption a7 Class Gene Cytoband Promoter CpG island * Representative mRNA ACC Representative protein ACC Subcellular location GSTs Alpha (a) GSTA1 6p12 no NM_145740 NP_665683 cytoplasm GSTA2 6p12.1 no NM_000846 NP_000837 cytoplasm GSTA3 6p12 no NM_000847 NP_000838 cytoplasm GSTA4 6p12 yes NM_001512 NP_001503 cytoplasm GSTA5 6p12.1 no NM_153699 NP_714543 cytoplasm (by similarity).

Techniques: Expressing, Quantitative RT-PCR, Methylation, DNA Methylation Assay

Journal:

Article Title: DNA hypermethylation regulates the expression of members of the Mu-class Glutathione-S-Transferases and Glutathione Peroxidases in Barrett's adenocarcinoma

doi: 10.1136/gut.2007.146290

Figure Lengend Snippet: Three esophageal cancer cell lines (OE33, SKGT4, and TE7) were treated with 2 μM 5-Aza for 72 hours and/or 100 nM TSA for 24 hours. The methylation levels were determined by pyrosequencing. The mRNA expression was calculated as the relative expression as compared to reference gene (HPRT) using the formula 2(Et-Rt) where Et is the threshold cycle number experimental gene in the test sample and Rt is the threshold cycle number for the reference gene in the test sample. The results were multiplied by 100 for a better visualization. The average percentage of DNA methylation of each gene is shown on the left side; whereas the relative mRNA expression of each gene is shown on the right side.

Article Snippet: The remaining 14 genes with CpG islands were analyzed by qRT-PCR and quantitative bisulfite pyrosequencing analysis of promoter DNA methylation. table ft1 table-wrap mode="anchored" t5 caption a7 Class Gene Cytoband Promoter CpG island * Representative mRNA ACC Representative protein ACC Subcellular location GSTs Alpha (a) GSTA1 6p12 no NM_145740 NP_665683 cytoplasm GSTA2 6p12.1 no NM_000846 NP_000837 cytoplasm GSTA3 6p12 no NM_000847 NP_000838 cytoplasm GSTA4 6p12 yes NM_001512 NP_001503 cytoplasm GSTA5 6p12.1 no NM_153699 NP_714543 cytoplasm (by similarity).

Techniques: Methylation, Expressing, DNA Methylation Assay