Global DNA Methylation Analysis

Genomic DNA was purified from tumor, lung, spleen, and liver cells using High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany). Accordingly, 40 mg tissue samples were lysed at 70°C with proteinase K followed by DNA binding, washing, and eluting according to the instructions of manufacturer. Global DNA methylation was evaluated using the MethylFlash Methylated DNA Quantification Kit (Epigentek, Farmingdale, New York) for analyzing the levels of 5-methylcytosine (5-mC). Purified DNA samples (100 ng) were incubated in 96-well plates coated with 5-mC antibody at 37°C for 90 minutes. After washing, a capture antibody was loaded onto the plates and incubated at room temperature for 30 minutes. Thereafter, the plates were washed and an enhancer solution was added and incubated for 30 minutes at room temperature. The percentage of methylated DNA was proportional to the optical density at 450 nm. DNA methylation was estimated using positive control (PC; 50% 5 mC) and negative control (NC; 50% unmethylated cytosin) controls according to Equation 2

where S is the amount of input DNA (100 ng) and P is the amount of input PC (5 ng). The levels of methylated DNA are calculated as percentage of the total DNA. More information are available in previous studies.^9,10