Methylation specific polymerase chain reaction

MSP was performed to evaluate the methylation status of the BAX and BCL2 promoters. MSP-specific primers are able to discern between methylated and unmethylated DNA sequences. The designing of MSP primers was done through MethPrimer software (17). The forward and reverse primers for BAX and BCL2 genes have been listed (Table 1).

For the MSP test, the bisulfite-treated DNA of MOLT-4 cells was used along with control samples including SssItreated DNA, meth primer positive control, peripheral blood SBS-treated DNA, a positive control for unmethylated DNA-specific primer (unmeth primer), normal human DNA with no SBS treatment as the negative control for both primers, and No-DNA sample as a negative control for the PCR reaction. The PCR mixture contained 12.5 µL of PCR master mix (Ampliqon, DENMARK), 1.25 µL of each forward and reverse primers (in a final concentration of 0.5 pM), bisulfite-modified DNA (150 ng), and double distilled water, in the final volume of 25 µl. The thermal cycler (MyCycler-BioRad) was used for amplification. The time and thermal periods of PCR were as follows: 5 minutes at 95°C for 1 cycle, subsequently 45 seconds at 95°C, 30 seconds at 59°C, then 45 seconds at 72°C, for 35 cycles. Next, an ultimate extension for 5 minutes at 72°C. Finally, in order to separate the PCR reaction products, electrophoresis was carried out on a 1.5% agarose gel and colored using safe stain, then studied under UV light.