Quantitative methylation-specific PCR (qMSP) assay

Primer sequences were designed based on the region of the TNFRSF10C and TNFRSF10D genes rich in CG sites. The primer sequences were as follows: TNFRSF10C forward, 5′-AGGGTGCGATTTAGGATTTAG-3′ and reverse, 5′-CGATAACGACGACGAACTT5′; TNFRSF10D forward, 5′-CGACGATGAAGACGACGAAT-3′ and reverse, 5′-AAACCAAACCATAACTCCTAAACC-3′. The conditions for qMSP were as follows: Initial denaturation at 95°C for 10 min, denaturation at 95°C for 20 sec, annealing at 56°C for 20 sec and extension at 72°C for 30 sec for 45 cycles. The melting curve program included 95°C for 15 sec, 60°C for 60 sec and then from 60°C to 95°C at a speed of 0.11°C/sec.

DNA was extracted from patient tissue samples according to the instructions of the QIAamp DNA FFPE Tissue kit (Qiagen GmbH). The extracted samples were measured for purity and concentration using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.). The DNA concentration of all samples was >25 ng/µl, and the purity of DNA was 1.8–2.0 based on the absorbance ratio at 260 and 280 nm. The DNA was subjected to bisulfite modification using the EZ DNA Methylation-Gold™ kit according to the manufacturer's instructions (Zymo Research Corp.). The primers and the bisulfite-modified DNA were then subjected to qMSP. The total reaction system was 10 µl, containing 5 µl SYBR mixture, 4 µl double-distilled H₂O, 0.5 µl each of the forward and reverse primers and 0.5 µl modified DNA. To avoid errors in sample loading, ACTB was used as an internal reference for each sample. The primer sequences of ACTB were as follows: Forward, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and reverse, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′. A total of 100% M-Sss I (New England BioLabs, Inc.) treated sperm DNA was used as the positive control and nuclease-free water was used as the negative control for each group. The qMSP product was then subjected to Qsep100 DNA fragment analysis (BiOptic, Inc.) and visualized. Three randomly picked qMSP products of each gene were Sanger sequenced for validation.

ACTB is a housekeeping gene that is often stably expressed in most healthy and tumor cells; thus, it is commonly used as an internal reference for quantitation of mRNA expression levels and qMSP assays (23). Completely methylated DNA referred to human sperm DNA treated with methylated-SssI, which allowed all C sites in the DNA to be methylated, including CG sites, and thus was used as a positive control. The formula for quantifying methylation levels was used to calculate the relative methylation level by the comparison between the target gene and the ACTB gene (24,25). For each sample, the relative methylation value was determined using the 2^−∆∆Cq method. The sample methylation reference percentage (PMR) was calculated using the following formula: PMR=2^−∆∆Cq ×100%; ∆∆Cq=sample DNA (Cq_gene-Cq_ACTB)-fully methylated DNA (Cq_gene-Cq_ACTB).