Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime

Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT-qPCR). Currently, there is no alternative to RT(-) controls to evaluate the impact of the gDNA background on RT-PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(-) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a nontranscribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing similar to 60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(-) controls and accurately corrects for signals derived from gDNA in RT-qPCR.