The cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, and incubated in 5% CO2 at 37°C. A 68-year-old woman with chronic hepatitis C was diagnosed with HCC in the right lobe and underwent liver resection. Specimens of her tumor and adjacent non-tumorous tissues were excised, and total RNA and DNA were extracted. Total RNA was sent to the manufacturer of Affymetrix to prepare it for expression array analysis. Genomic DNA was used for KU55933 mw a SNP-Chip array, and bisulfite-converted DNA was used for the Ilumina Infinium HumanMethylation 27 BeadChip (Illumina, San Diego, CA, USA). The

tumor was pathologically confirmed as HCC. RNA and DNA of tumor samples were extracted from an area consisting of >80% cancerous cells. HCC tissue (HTs) Selleckchem MK-8931 and normal tissue (NTs) samples were obtained from 48 patients (43 males, five females) who underwent liver resection at Nagoya University Hospital, Nagoya, Japan between 1994 and 2001. The patients were aged from 39 to 77 years (mean ± SD, 62.4 ± 7.9 years). Thirty-eight patients had hepatitis C and seven had hepatitis B. The median duration of follow-up was 80.7 months (range 15.2–213.1 months). All tissues were reviewed pathologically to confirm the diagnosis of HCC. Written informed consent, as required by the institutional review

board, was obtained from all patients. The tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until required. Genomic DNA was obtained from the tissue samples by proteinase K digestion, followed by phenol/chloroform extraction. RNA isolation, microarray and gene chip affymetrix procedures The expression array and SNP array were performed, as previously described

[12–17], using total RNA and DNA extracted from the 68-year-old woman’s tissue samples. Methylation array platform The Illumina Infinium HumanMethylation27 BeadChip protocol requires 500 ng to 1 μg of bisulfite-converted DNA [26]. Of the approximately 28 million CpG sites found throughout the haploid human genome, Illumina initially designed Infinium methylation probes for 27,578 CpG sites located in promoter regions (up Bcl-w to 1 kb upstream or 500 bp downstream of the transcription start sites). Of these, 27,324 CpG sites relate to 14,475 consensus coding sequences, including around 1000 cancer-associated genes, and 254 CpG sites relate to approximately 100 micro-RNA genes. The probes were preferentially selected to occur within CpG islands using the NCBI “relaxed” definition of a CpG island: CpG islands identified bioinformatically with a CpG content of >50% and an observed/expected ratio of >0.6 [27]. Bisulfite-converted DNA is then whole-genome amplified, enzymatically fragmented, and hybridized to the array. During hybridization, the bisulfite-converted DNA anneals to methylation-specific probes on the chip.