MethyLight and Digital MethyLight.

MethyLight is a quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation of candidate regions of the genome. MethyLight is uniquely suited for detecting low-frequency methylated DNA regions against a high background of unmethylated DNA, as it combines methylation-specific priming with methylation-specific fluorescent probing. The quantitative accuracy of real-time PCR and the ability to design bisulfite-dependent, DNA methylation-independent control reactions together allow for a quantitative assessment of these low frequency methylation events. Here we describe the experimental steps of MethyLight analysis in detail. Furthermore, we present principles and design examples for three types of quality control reactions. QC-1 reactions are methylation-independent reactions to monitor sample quantity and integrity. QC-2 reactions are bisulfite-independent reactions to monitor recovery efficiencies of the bisulfite conversion methodology used. QC-3 reactions are bisulfite-independently primed reactions with variable bisulfite-dependent probing to monitor completeness of the sodium bisulfite treatment. We show that these control reactions perform as expected in a time course experiment interrupting sodium bisulfite conversion at various timepoints. Finally, we describe Digital MethyLight, in which MethyLight is combined with Digital PCR, for the highly sensitive detection of individual methylated molecules, with use in disease detection and screening.