Extraction ?l PCR mixture. Final reaction condition was 1x

Extraction and bisulfite conversion of the DNA from formalin-fixed and paraffin embedded (FFPE) samples FFPE tumor and normal samples from pancreatic cancer patients and FFPE normal samples from no pancreatic cancer patients were collected. A pathologist confirmed the normal sections from the hematoxylin and eosin (H & E) stained slides. Tissue sections were serially cut as 10 ?m thickness.  For extraction DNA from FFPE samples, tissues are subjected to xylene treatment and rehydrated using ethanol washes.  And then proteins are digested by proteinase K and digestion lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), at 55°C for 4 hours. Next, bisulfite conversion was performed using Zymo EZ DNA Methylation Kit, Zymo Research, following the manufacturer’s instructions.  Finally, bisulfite modified DNA was eluted by dH2O.DNA Methylation Analysis  Bisulfite treated DNA samples were analyzed for the methylation levels of 3 genes, CDO1, TAC1 and CHFR, using quantitative real-time Methylation Specific PCR (qMSP).  The qMSP levels were normalized against the respective values of the internal control gene ?-Actin. The primers and hybridization probes for qMSP were designed based on this sequence by using UCSC genomic browser web site and Primer3 (v.0.4.0) 1.  Primer sequences are 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 antisense primer, 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 Tris pH 8.8, 6.7 mM MgCl2 and 10mM -mercaptoethanol in a nuclease-free DI water solution.  Amplification reactions were performed using 96 well-plates (MicroAmp®) in triplicate. Thermocycling conditions were: 95°C for 5 min, 50 cycles at 95°C for 15 seconds, and 65°C for 1min and 72°C for 1 min.  The StepOnePlusTM Real-Time PCR System was used (Applied Bio Systems).Methylation values for each sample were calculated ?CT method: ?CT= CTsample – CT?-Actin. For replicates which were not detected, a CT of 100 was used, creating minimum methylation value of near 0.  The mean 2^-?CT value was calculated as follows: the methylation value = (2^-?CTrepricate1 + 2^-?CTrepricate2 + 2^-?CTrepricate3) / 3 1.  For the methylation value which was greater than 1, the value of 1 was used, creating max methylation value of 1.