Extraction and bisulfite conversion of the DNA from formalin-fixedand paraffin embedded (FFPE) samples FFPE tumor and normalsamples from pancreatic cancer patients and FFPE normal samples from nopancreatic cancer patients were collected. A pathologist confirmed the normalsections from the hematoxylin and eosin (H & E) stained slides. Tissuesections were serially cut as 10 mm thickness. For extractionDNA from FFPE samples, tissues are subjected to xylene treatment and rehydratedusing ethanol washes.
And then proteins aredigested by proteinase K and digestion lysis buffer, which contains denaturingagents such as sodium dodecyl sulfate (SDS), at 55°C for 4 hours. Next,bisulfite conversion was performed using Zymo EZ DNA Methylation Kit, ZymoResearch, following the manufacturer’s instructions. Finally, bisulfite modified DNA was eluted bydH2O.DNA Methylation Analysis Bisulfite treated DNAsamples were analyzed for the methylation levels of 3 genes, CDO1, TAC1 andCHFR, using quantitative real-time Methylation Specific PCR (qMSP). The qMSP levels were normalized against the respectivevalues of the internal control gene ?-Actin. The primers and hybridizationprobes for qMSP were designed based on this sequence by using UCSC genomicbrowser web site and Primer3 (v.
Primer sequencesare listed in Table 1. Two ?l of bisulfited DNA was added to 23 ?l PCR mixture.Final reaction condition was 1x buffer, 200 nM sense primer, 200 nM antisenseprimer, 80 nM probe, 10 nM of fluorescein reference dye (Life Technologies),0.167 mM dNTPs (VWRQuotation), and a single unit of Platinum Taq® DNA Polymerase(invitrogen). 1x buffer consisted of 16.6mM(NH4)2SO4, 67mM TrispH 8.
8, 6.7 mM MgCl2 and 10mM -mercaptoethanol in a nuclease-free DI watersolution. Amplification reactions wereperformed using 96 well-plates (MicroAmp®) intriplicate.
Thermocycling conditions were: 95°C for 5 min, 50 cycles at 95°Cfor 15 seconds, and 65°C for 1min and 72°C for 1 min. The StepOnePlusTMReal-Time PCR System was used (Applied Bio Systems).Methylation values for each sample were calculated DCT method: DCT= CTsample – CT?-Actin. For replicates which were not detected, a CT of 100 wasused, creating minimum methylation value of near 0. The mean 2^-DCTvalue was calculated as follows: the methylation value = (2^-DCTrepricate1 + 2^-DCTrepricate2 + 2^-DCTrepricate3) / 3 1. For the methylationvalue which was greater than 1, the value of 1 was used, creating maxmethylation value of 1.