A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction

DNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference. We determined DNA methylation levels in several promoter regions in two setups implementing different references: mock-digested and treated with a restriction enzyme that has no recognition sites within examined amplicons. Fragmentation of reference templates allowed removing the overestimation effect, thereby improving measurement accuracy. •CpG methylation is one of key mechanisms of epigenetic regulation in vertebrates.•CpG methylation sensitive endonucleases are used to estimate DNA methylation.•qPCR of DNA protected from digestion by CpG methylation is often greatly overstated.•Restriction enzyme digestion of template DNA removes the overestimation effect.•Fragmentation of genomic DNA improves PCR performance and quantitation accuracy.