Quantifying DNA Methylation by qMSP

For measuring DNA methylation, DNA was bisulphite-modified (resulting in deamination of unmethylated cytosine to uracil) using an EZ DNA methylation^TM kit (ZYMO Research, Irvine, CA, USA), according to the manufacturer’s protocol, and subjected to quantitative methylation-specific PCR (qMSP), as previously described53 (link). PCR qMSP amplification was performed using the StepOne Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA, USA), with ACTB used to normalize the input DNA. The absolute amount of methylated NR4A3 was deter ed by the threshold PCR cycle number (Ct), for each sample, using a standard curve generated by qMSP of an SssI-treated cloned DNA fragment. The relative percentage of NR4A3 methylation was calculated as the NR4A3 vs ACTB ratio for each sample, divided by the same ratio for SssI-treated sperm DNA (positive DNA methylation control, Millipore, Billerica, MA, USA) and multiplied by 100. Experiments were repeated twice.