Global DNA methylation assay

The concentration of 5-methylcytosine (5-mC) in gDNA was quantified using 5-mC DNA ELISA kit and was measured according to the manufacturer’s instructions (Zymo Research, Irvine, CA). Briefly, standard DNA used to establish the standard curve was prepared by mixing negative (unmethylated DNA at 100 ng/μl) and positive (completely methylated DNA at 100 ng/µl) controls at different proportions. Standard or sample gDNA of 100 ng each was mixed with 5-mC coating buffer up to 100 μl and denatured at 98°C for 5 minutes followed by quick transfer onto ice for 10 minutes. Denatured gDNA was coated onto 96-well plates by incubation at 37°C for 1 hour. Following 3 washes with 5-mC ELISA buffer, 200 μl of 5-mC ELISA buffer was added for another incubation at 37°C for 30 minutes. An antibody mixture consisting of anti-5-mC and secondary antibody in 5-mC ELISA buffer in the ratio of 1:2:2000 in 100 µl was added for incubation at 37°C for 1 hour. After washing, 100 µl HRP developer was added, and color development was achieved by incubation at room temperature for 1 hour. Then the absorbance at 450 nm was obtained from each reaction on a plate reader (BioTek Epoch 2, BioTek/Agilent, Santa Clara, CA) for the establishment of a standard curve and for the quantitative analyses of samples from the curve. The results were validated in triplicate for each standard and sample.