Fig. 2

A DNA integrity after bisulfite conversion: Pre-amplification visualization on agarose gel electrophoresis. Two µl of the eluted bisulfite-converted DNA was loaded and run on 2% gel in TBS buffer for 60 min. Smears with equal intensities indicate the presence of the converted DNA. Lanes (1–3) good conversion, lanes (5 and 6) weak conversion, and lanes (4 and 7) no conversion. B The specificity of the primers. Bisulfite Modified (BM-DNA) and Unmodified DNA (UM-DNA) were amplified with primers specific for unmethylated (U) and methylated (M) DNA. Only modified DNA showed PCR amplicons. GAPDH of the UM-DNA used as a control for equal loading. C Localization of methylated and unmethylated PCR band of every TSG. Ethidium bromide-stained agarose gel electrophoresis of bisulfite modified DNA amplified with primers specific for unmethylated (U) and methylated (M) DNA. Lane 1 100 bp molecular marker, lane 2–3 PCR amplicons of RASSF1A promoter detected at 203 bp, lane 4-5 PCR amplicons of CHFR promoter at 144 bp, lane 6-7 PCR amplicons of GSTP1 promoter at 159 bp, lane 8-9 PCR amplicons of MGMT promoter detected at 93 bp, lane 10-11 PCR amplicons of hMLH1 promoter detected at 154 bp. GAPDH of the UM-DNA used as a control for equal loading. D MSP assay interpretation: Representative experiment showing molecular ladder marker, positive and negative controls (lanes 1–4), and bisulfite modified DNA amplified with primers specific for unmethylated (U) and methylated (M) DNA (lanes 5–10). Methylated promoter of a gene contains only the methylated amplicon (lane 5–6), the partially methylated promoter contains both methylated and unmethylated amplicons (lane 7–8), and the unmethylated promoter contains only the unmethylated amplicon (lane 9–10). (+ ve con.) positive control, (-ve con) negative control. M Marker, L 100 bp ladder. GAPDH of the UM-DNA was used as a control for equal loading. E Heat map of the TSGs promoter methylation in the HCC and ANTs. The intensity of the PCR bands reflecting the methylation status of the TSGs promoter in HCC and ATC were plotted in a heat map. Red and green colors represent relative high and low TSGs methylation values, respectively