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 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.
Inserm/Université Paul Sabatier UMR1048, Institut des Maladies Métaboliques et Cardiovasculaires, BP84225, 31432 Toulouse cedex 4, France. henrik.laurell@inserm.fr
Henrik
H
Laurell
Jason S
JS
Iacovoni
Anne
A
Abot
David
D
Svec
Jean-José
JJ
Maoret
Jean-François
JF
Arnal
Mikael
M
Kubista
D000818:Animals; D004247:DNA / Q000032:analysis; D059018:DNA Contamination; D017931:DNA Primers; D023281:Genomics; D051379:Mice; D008810:Mice, Inbred C57BL; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004247:DNA