Methylation-specific droplet digital PCR (MS-ddPCR) for detection and absolute quantification of rare methylated alleles

Abstract The recent advent of Droplet Digital PCR (ddPCR) provides an unprecedented opportunity for DNA methylation studies due to its enhanced sensitivity and precision over conventional PCR-based methods. Here, we provide a detailed protocol for the design and implementation of methylation-specific droplet digital PCR (MS-ddPCR) assays using Bio-Rad's QX200 system. We focus on the experimental considerations for detection and absolute quantification of rare methylated events and present an example of a successful assay for detecting DNA methylation in the promoter region of mir342/EVL, a potential DNA methylation biomarker for colon cancer risk. We also discuss the common technical difficulties and troubleshooting processes for conducting a successful MS-ddPCR experiment.