MspI and HpaII is a pair of restriction enzymes that recognize the same DNA sequence 5’-CCGG, but exhibit different sensitivity to DNA methylation. The fragment number produced by Hap II restriction enzyme is dependent on the methylation status of restriction site in whole genome, however the fragment number produced by Msp I is stable and give a constant CT value in qPCR, so the fragment ratio between Hap II and Msp I library made with NanoMeth® Kit is the global un-methylation percentage, which can be calculate by the following equation: