Abstract
Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.
Highlights
Data from Quantitative PCR (qPCR) experiments are taken within each enzymatic reaction curve at the quantification cycle (Cq)
Thereby, Droplet Digital PCR (ddPCR) technology can be used for extremely low-target quantitation from variably contaminated samples where the sample dilution requirements to assure consistent and acceptable reaction efficiency, primer annealing and Cq values for qPCR would likely lead to undetectable target levels[11, 15]
Since qPCR is a sample interdependent technology where the relative quantity and normalized gene expression data rely on ΔCq values, the analysis from a single plate assures the best quality results by eliminating any bias from inter-plate variability[16, 17]
Summary
Data from qPCR experiments are taken within each enzymatic reaction curve at the quantification cycle (Cq). There are two distinct differences: 1) the partitioning of the PCR reaction into thousands of individual reaction vessels prior to amplification and 2) the acquisition of data at reaction end point These factors offer the advantage of direct and independent quantification of DNA without standard curves giving more precise and reproducible data versus qPCR especially in the presence of sample contaminants that can partially inhibit Taq polymerase and/or primer www.nature.com/scientificreports/. Thereby, ddPCR technology can be used for extremely low-target quantitation from variably contaminated samples where the sample dilution requirements to assure consistent and acceptable reaction efficiency, primer annealing and Cq values for qPCR would likely lead to undetectable target levels[11, 15]. In samples with low concentrations of nucleic acids and variable amounts of Taq inhibitors, ddPCR technology was shown to convert uninterpretable results generated from qPCR to highly quantitative and reproducible data
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