Bisulfite sequencing of TFPI-2 in normal oral and OSCC tissues. (A) The region centering on the TFPI-2 transcription start site, comprising 31 CpG sites. The distribution of CpG sites (vertical bar) and the position of the hybridized PCR primer pairs for bisulfite sequencing (BS) and quantitative methylation-specific PCR (qMSP) are shown. Two CpG sites in the qMSP forward primer are indicated (filled triangles), and the 11 CpG sites analyzed by pyrosequencing assay are boxed. (B) Representative bisulfite sequencing assay for normal oral and OSCC tissues. For each tissue sample, five randomly chosen clones were sequenced and the methylation status of each of the 31 CpG sites is indicated by the circles: closed and open circles represent methylated and unmethylated CpG sites, respectively.