Laurell, H., Iacovoni, J., Abot, A., Svec, D., Maoret, J.-J., Arnal, J.-F., Kubista, M. Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime. Nucleic Acids Research, 40: 1-10, 2012. ISSN 0305-1048.
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 non-transcribed 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 ∼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.