Henrik Laurell1, Jason Iacovoni1, Jean-José Maoret1, Jean-François Arnal1, Mikael Kubista2
1Inserm / Université Paul Sabatier UMR1048, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC); 2TATAA Biocenter AB, Göteborg, Sweden
Genomic DNA (gDNA) contamination is an inherent problem during RNA purification, which can lead to non-specific amplification and aberrant results in reverse transcription (RT)-qPCR. gDNA sensitivity for most qPCR assays can be greatly diminished by appropriate precautions during assay design. However, regardless of the primer design strategy, the inability of a Gene-Of-Interest (GOI) assay to amplify gDNA needs to be validated experimentally. ValidPrime offers this possibility. Prior to ValidPrime, RT-minus controls were the only option available to evaluate the impact of the gDNA background on RT-PCR data. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA), targeting a non-transcribed locus, and gDNA reference sample(s) that permit normalization for GOI-specific differences in gDNA sensitivity. The RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for gDNA, up to 60% of the total signal, while substantially reducing the number of required qPCR control reactions.
In conclusion, ValidPrime 1) offers a cost-efficient alternative to RT(-) controls; 2) accurately validates qPCR assays in terms of their sensitivity to gDNA 3) allows correction for gDNA-derived signals; 4) reduces the need for DNase treatment 5) The dedicated ValidPrime software greatly simplifies the analysis.
Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime. Laurell H, Iacovoni JS, Abot A, Svec D, Maoret JJ, Arnal JF, Kubista M. Nucleic Acids Res. 2012 April 1;40(7):e51.
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