I am trying to quantify 5mC and 5hmC using Epimark 5mC and 5hmC kit from NEB. I did the end-point PCR after glucosylation and digestion with MspI and HpaII. It didnt generate a a yes or no answer, which is possible if the position is partially methylated or hydroxymethylated (which might have happened in the studied locus). I did qRT-PCR and follow the formulae mentioned in the protocol to calculated the percentage of methylation, to which I got negative values in some cases and the total methylation (sum of percentage of 5mc, 5hmC and unmethylated) is not equal to 1. The experiment was done in triplicates with case and control and normalized as mentioned in the protocol. Where am I getting it wrong? Even if I ignore the negative value for percentage, the total methylation value is not equal to 1. Will appreciate any help or suggestion.

Below is the link to the protocol.

https://www.neb.com/protocols/0001/01/01/reaction-protocol-for-epimark-5-hmc-and-5-mc-analysis-kit-e3317

Thanks