DNA extraction, bisulfite modification and methylation- specific PCR (MS-PCR)

Genomic DNA was isolated from the cells and tissues using a Takara Mini BEST Universal Genomic DNA Extraction kit (Takara) according to the manufacturer's instructions. DNA modification was performed as previously described (23). A total of 500 ng of the extracted DNA was bisulfite-modified with the EZ DNA Methylation-Gold™ kit (Zymo Research). Modified DNA templates were used for MS-PCR with Zymo TaqTM PreMix (E2003; Zymo Research) following the instructions of the manufacturer. The online software MethPrimer (http://www.urogene.org/methprimer/) profiled CpG islands in the region that is located from −2,000 to −200 bp upstream of ATG in the HAVCR2/LGALS9 promoters. One pair of primers was designed to amplify the HAVCR2/LGALS9 promoter regions. The primer pairs used for MS-PCR are listed in Table II. The thermocycling conditions were as follows: 95°C for 10 min and 40 cycles of 95°C for 30 sec. The annealing temperature for the methylated primer pairs of HAVCR2 and LGALS9 was 60°C, respectively, while that for the unmethylated primer pairs was 55°C and 56.3°C, respectively for 30 sec and 72°C for 30 sec, followed by an incubation step at 72°C for 7 min. The MS-PCR products were separated on a 2% agarose gel, stained with Gelview and visualized under ultraviolet illumination (Bio-Rad). Methylation level was calculated by the ratio of methylated and unmethylated levels. The calculation method was as follows: Methylation(M)=M/(M + U); unmethylation=U/(M + U). The grey value of each band represented its relative expression and was measured using ImageJ software. Each reaction was performed in triplicate.