MethylQuant: A Real-Time PCR-Based Method to Quantify DNA Methylation at Single Specific Cytosines

MethylQuant is a cost-effective and relatively simple technique which enables quantitative analysis of the methylation status of a single cytosine at specific positions in DNA that can be assimilated to the quantitative detection of a single nucleotide polymorphism (SNP). After bisulfite conversion of DNA and PCR amplification of the region of interest, the methylation status is quantified by methylation-specific real-time PCR with one of the primers harboring the methylation status-specific nucleotide at the most 3′ end. In parallel, the amount of amplifiable DNA is quantified by a methylation-independent real-time PCR. In this protocol, we describe in detail the different stages of the MethylQuant procedure and discuss the parameters of DNA bisulfite conversion and quantitative PCR analysis with SYBR green that are crucial to achieve an accurate quantification of the methylation status of a particular cytosine. The practical aspects of DNA bisulfite conversion, primer design, and quantitative PCR analysis, discussed hereafter, should be of general interest even outside the context of the MethylQuant technique.