Transitioning Your Assay From Quantitative PCR To Droplet Digital PCR

By Dianna Maar and Andrew M Prantner, Bio-Rad Laboratories, Inc.,

This application note will discuss migrating assays from real-time quantitative PCR (qPCR) to Droplet Digital PCR (ddPCR). These two PCR methods determine the concentration of sample DNA using fundamentally different approaches and a bridging study between the two methods presents data demonstrating the differences and similarities to consider. In qPCR, concentration measurements from the bulk PCR reaction are relative to a control or standard DNA material because the fluorescent signal is based on the proportionality between fluorescence and DNA amount. In Droplet Digital PCR, DNA concentration is determined directly by partitioning the PCR reaction into thousands of individual droplets that are counted as positive or negative and evaluated with Poisson statistics after PCR amplification is complete. Transitioning from quantitative PCR to Droplet Digital PCR can be simple and provide consistent, robust ddPCR results that can be directly compared to past qPCR data by conducting a straightforward bridging study.