Bisulfite PCR and efficiency of bisulfite modification

Twenty-four hours after siRNA transfection, DNA was extracted from bEnd.3 cells with NucleoSpin Tissue kit (Macherey-Nagel, Germany). Bisulfite conversion was performed using EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA). CpG island prediction and methylation primer design were performed using MethPrimer²⁶. PCR was carried out in a final volume of 50 μl containing 100 ng bisulfite-treated DNA, 2.5 mM MgCl₂, 400 nmol each primer, 0.3 mM each dNTP, 0.25 μl TaKaRa EpiTaq^™ HS (for bisulfite-treated DNA) (1.25 U/50 μl). PCR cycling conditions were as follows: 40 cycles of 10 s at 98 °C, 30 s at 56 °C for, 90 s at 72 °C and finally ended with 1 min at 72 °C for an extension. PCR products were separated on 1.0% agarose gel, purified using QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), and then cloned into the pMD20-T vector using Mighty TA-cloning Kit (Takara, Japan). At least ten positive recombinant colonies of each product were sequenced by the Sanger method (Eurofins Genomics, Japan). The analysis of 65 bisulfite sequences was carried out with the QUMA (QUantification tool for Methylation Analysis) program (http://quma.cdb.riken.jp).