Do I need an internal methylated DNA reference?
No. For any given target sequence, the same primer pair is used to amplify the four different digests allowing the PCR results to be reliably and directly compared. In contrast, bisulfite conversion-based PCR methods use two different pairs of primers to amplify either methylated or unmethylated templates of a given target sequence after conversion. Differences in their amplification efficiencies could cause biased results, therefore requires normalization to in vitro methylated reference DNA. However, since the primers need similar amplification efficiencies and the methylation need to be consistently complete, reliable results can only be achieved after careful trial and error based optimization and additional cost as well.
