Methylation-Specific PCR Protocol for DNA Analysis

Each methylation-specific PCR (MSP) reaction was performed using 2 μL DNA template in a 20 μL reaction volume containing 1 μL each of forward and reverse primers (concentration 10 μmol/L), which was designed using MethPrimers^{[24 (link)]} (Table 1), 0.4 μL MSP DNA Polymerase, 1.7 μL 10× MSP PCR Buffer, and 1.4 μL dNTPs (Tiangen Biotech, Beijing, China). The PCR reaction conditions were as follows: denaturation at 95°C for 5 min → amplification at (94°C 20 s, 57°C 30 s, 72°C 20 s) for 35 cycles → extension at 72°C for 5 minutes. The reaction was set up with a blank control group (ddH₂O group), a water blank, a methylated sample blank, an unmethylated sample blank as a negative control, a methylated control, and an unmethylated control (Tiangen Biotech). Then the PCR products were analyzed by 1% agarose gel electrophoresis (DYCP-32B, Liuyi, Beijing China) and photographed using the Tianneng UV system (Tanon-5200 Multi; Tanon Science and Technology Co. Ltd., Shanghai China). There was a clearly visible band in the gel of methylated samples, when using the methylated primers. MSP results were verified by the bisulfite sequencing PCR (BSP) assay, which was performed by a professional sequencing company (Genechem, Shanghai China).