Genome-wide DNA methylation analysis of pancreatic tissue samples obtained from patients with pancreatic ductal adenocarcinoma.

Genome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip in 34 samples of non-cancerous pancreatic tissue and 82 samples of cancerous tissue samples obtained from 82 patients with pancreatic ductal adenocarcinoma. From fresh frozen tissue samples, high-molecular-weight DNA was extracted using phenol-chloroform followed by dialysis. Five-hundred nanogram of genomic DNA was subjected to bisulfite treatment using an EpiTect Bisulfite Kit (QIAGEN), in accordance with the manufacturer’s protocol. After bisulfite treatment, DNA methylation status at 485,764 probe sites was examined at single-CpG resolution using the Infinium HumanMethylation450K BeadChip (Illumina, San Diego, CA), according to the manufacturer’s instructions. Hybridization fluorescence signals were read by the iScan reader (Illumina). The data were assembled using GenomeStudio methylation software (Illumina). At each CpG site, the ratio of the fluorescent signal was measured using a methylated probe relative to the sum of the methylated and unmethylated probes, i.e. the so-called β-value, which ranges from 0.00 to 1.00, representing the fully unmethylated and fully methylated values of an individual CpG site, respectively.