Following good laboratory practice any experiment needs to be properly controlled.

In PCR based methylation screening experiments two controls are necessary: methylated (positive) and non-methylated (negative).

Those controls are normally chemically modified DNA templates consisting of methylated and non-methylated versions of the locus of interest and are reference points for the analyses of an unknown sample.

In the Methylation-Sensitive High-Resolution Melting (MS-HRM) protocol methylation status of a screened sample is assessed by comparison of the melting profile (see also: What is DNA melting?)  of the PCR product amplified from a screened sample (Figure 1 – green) with the melting profiles of PCR products amplified from methylated (Figure 1 – blue) and non-methylated (Figure 1 – red) controls.

In principle, hypomethylation is observed if the melting profile of a screened sample does not overlap with the methylation profile of the positive control and hypermethylation in observed when the melting profile of the sample does not overlap with the melting profile of a non-methylated control.

Figure 1: