For each analysis, the bisulfite conversion was performed with 500 ng provided genomic DNA using the EZ DNA methylation kit (ZymoResearch, Inc., CA). The PCR reaction was performed based on recommended assay conditions (EpigenDx, MA) using 0.2 μM of each primer with one of the PCR primers being biotinylated in order to purify the final PCR product using Sepharose beads. The PCR product was bound to Streptavidin Sepharose HP (Amersham Biosciences, Uppsala, Sweden), and the Sepharose beads containing the immobilized PCR product were purified, washed and denatured using 0.2 M NaOH solution and rewashed using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing, Qiagen) as recommended by the manufacturer. 10 μl of the PCR products were sequenced by Pyrosequencing PSQ96 HS System (Pyrosequencing, Qiagen) following the manufacturer’s instructions (Pyrosequencing, Qiagen). The methylation status of each CpG site was analyzed individually as an artificial T/C SNP using QCpG software (Pyrosequencing, Qiagen).
