ELISA-based global DNA methylation quantification

The percentage of methylation in total DNA was determined by measuring 5-methylcytosine (5-mC) using a 5-mC DNA ELISA kit (Zymo Research Corp, Orange, CA, USA) that features a unique anti-5-mC monoclonal antibody that is both sensitive and specific for 5-mC. Assays were performed according to the manufacturer’s instructions with the modified standard curve. The percentages of prepared methylated DNA standards were 5, 1.66, 0.55, and 0.185%. The amount of total DNA used was 100 ng. Logarithmic second-order regression was used to calculate the results. Since the positive and negative controls used to prepare the standard curve consist of Escherichia coli gDNA, the obtained results were multiplied by 5.68 to correct for fold differences in the density of CpGs between E. coli and humans following the recommendations from the manufacturer.