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

Abstract : 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.
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Contributor : Marie Francoise Simon Connect in order to contact the contributor
Submitted on : Friday, September 14, 2012 - 5:27:01 PM
Last modification on : Monday, July 4, 2022 - 9:33:26 AM
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Henrik Laurell, Jason Iacovoni, Anne Abot, David Svec, Jean-José Maoret, et al.. Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime.. Nucleic Acids Research, Oxford University Press, 2012, 40 (7), pp.e51. ⟨10.1093/nar/gkr1259⟩. ⟨inserm-00732321⟩



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