TAP-1 promoter methylation-specific PCR (MSP) assay

The TAP-1 promoter MSP assay was performed as previously described.35 Briefly, each reaction mixture comprised HotStarTaq Master Mix (Qiagen), forward and reverse primers targeting the TAP-1 promoter, RNase‐free water, and bisulfate-converted DNA. The MSP primer sequences are as follows: methylated forward, 5′-TTTTTTAAATGGTTGAGTTTTTCGT-3′, and reverse, 5′-TAAAACCTAAAACTCCGAATACCG-3′; unmethylated forward, 5′-TTTTTTAAATGGTTGAGTTTTTTGT-3′, and reverse, 5′-AAAACCTAAAACTCCAAATACCACC-3′. Reactions were started by heating to 95°C for 5 min, followed by 35 cycles of 95°C for 45 s, 57°C for 30 s and 72°C for 30 s, and a final extension for 10 min at 72°C. CpGenome Universal Methylated DNA (Sigma-Aldrich) and CpGenome Universal Unmethylated DNA (Sigma-Aldrich) kits were used as positive controls for methylated and unmethylated DNAs, respectively, whereas Ultrapure distilled water (Invitrogen) served as the negative control. The MSP products were separated on a 2% agarose gel and visualized using ethidium bromide staining with ultraviolet light transillumination.