Genomic DNA extraction Genomic DNA was extracted using the QIAamp DNA Mini Kit according to the manufacturers recommendations. Tissue samples were ground up by 3 mm diameter punches and then mixed with 700 uL lysis buffer containing 20 ug mL Labo Pass protease K, 20 mM TrisHCl, 5 mM EDTA, 400 mM NaCl, and 1% SDS solution. The mixed samples were incubated at 42 C overnight. After incubation, genomic DNA was purified by phenol chloroform extraction. Genomic DNA was eluted in 100 uL of water and quantified with a Nano Drop ND 100 device. Sodium bisulfite DNA modification Two micrograms of genomic DNA in a volume of 20 uL RNase free water was bisulfite converted using the Epi Tect fast DNA bisulfite kit. Bisulfite conversion was performed according to the manufacturers recom mendations.

The reaction was performed by mixing 85 uL bisulfite mix solution and 35 uL DNA protect buf selleck CORM-3 fer in 200 uL PCR tubes at room temperature. The bisulfite converted genomic DNA was eluted from the column with 100 uL dH2O and stored at 80 C until use. Methylation bead chip array Human Methylation 27 DNA Analysis Bead Chip is a methylation profiling tech nology based on bisulfite modification of DNA. This bead chip array can provide methylation information at a single base resolution for 27,578 CpG sites spanning more than 14,000 genes. One microgram of bisulfite converted genomic DNA was applied to the bead chips using Illumina supplied reagents and conditions. After exten sion, the array was fluorescently stained and scanned, and the intensities of the M and U bead types were measured.

Each methylation data point is represented by fluorescent signals from the M and U alleles. The ratio a total noob of fluorescent signals was then computed from the two alleles, B value. The B value reflects the methylation level of each CpG site. A B value of 0 1. 0 indicates the percent methylation from 0% to 100%, respectively. Quantitative methylation specific PCR Quantitative methylation status in the bisulfite converted genomic DNA was confirmed by quantitative real time PCR using the 7000 HT Real Time PCR System according to the manufacturers recommen dations. Methylation primers for 21 candidate genes and 18 CIMP markers were designed using the MethPrimer software. Primers for QMSP were designed for large promoter CpG islands containing detected CpG sites near the transcription start site. PCR reactions were per formed using an optical 96 well tray in a final volume of 20 uL. The reaction mixture consisted of 5 uL 2X Maxima SYBR Green ROX qPCR master mix, 250 nM of each primer, and 30 ng of bisulfite converted DNA template. The QMSP program was as follows, 50 C for 2 min and 95 C for 10 min, followed by 45 cycles at 95 C for 15 s, and then 60 C for 1 min.