Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution

This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis.