Validation of measuring DNA methylation with real-time PCR.

Bisulfite treatment changes cytosine to uracil only if it is not methylated (A). Differences in methylation are thereby converted into differences in sequence that can be detected by quantitative real-time PCR. Because the bisulfite conversion itself is not quantitative, primers have to distinguish between converted and nonconverted DNA (examples shown in B). In addition, primers used as internal standards (ACTB and MYOD1; Eads et al26) have to be independent of methylation, whereas primers localized in CpG islands have to be methylation sensitive and should give no signal on DNA methylated with SSS1-methylase (B).