MethyLight.

MethyLight is a sodium-bisulfite-dependent, quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation in genomic DNA. MethyLight relies on methylation-specific priming combined with methylation-specific fluorescent probing. This combination of methylation-specific detection principles results in a highly methylation-specific detection technology, with an accompanying ability to sensitively detect very low frequencies of hypermethylated alleles. The high sensitivity and specificity of MethyLight make it uniquely well suited for detection of low-frequency DNA methylation biomarkers as evidence of disease. At the same time, the quantitative accuracy of real-time PCR and the flexibility to design bisulfite-dependent, methylation-independent control reactions allows for a quantitative assessment of these low-frequency methylation events. We describe the experimental steps of MethyLight analysis in detail. Furthermore, we present here 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-independent 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.