Quantitative DNA Methylation Analysis

Bisulfite pyrosequencing was performed by EpigenDx laboratories LLC using their established protocol^{59 (link), 77 –79 (link)}. Briefly, 500ng of extracted DNA was bisulfite treated by EpigenDx using a proprietary bisulfite salt solution. Bisulfite treated DNA was purified using Zymogen DNA columns. For SNP mutation analysis, 5 ng of genomic DNA was used for PCR. The PCR was performed with 0.2 μM of each primer and one of the PCR primers is biotinylated to purify the final PCR product using Sepharose beads. The PCR product was bound to Streptavidin Sepharose HP (GE Healthcare Life Sciences), and purified, washed and denatured using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing, Qiagen). PCR products were sequenced by Pyrosequencing PSQ96 HS System (Pyrosequencing, Qiagen) following manufacturer’s instructions (Pyrosequencing, Qiagen). The methylation status of each locus was analyzed individually as a T/C SNP using QCpG software (Pyrosequencing, Qiagen).