ValidPrime® – Detect and Control gDNA Contamination in RT-qPCR

ValidPrime® is a unique qPCR-based assay developed to monitor and detect contaminating genomic DNA (gDNA) in RNA samples used for RT-qPCR. Unlike traditional controls, ValidPrime does not rely on reverse transcriptase or exogenous spike-ins. Instead, it detects non-transcribed pseudogenes, providing a robust, accurate measure of gDNA contamination within the biological sample itself.

Developed in collaboration with leading RT-qPCR researchers, ValidPrime enhances confidence in gene expression data, particularly in regulated or clinical workflows where RNA integrity and purity are critical.

ValidPrime® replaces the need to perform no reverse transcriptase (RT(-)) controls to test for the presence of genomic DNA (gDNA) for all samples in your real-time quantitative PCR (qPCR) profiling. In an expression profiling experiment based on m samples and n assays, traditional set up requires m RT(-) reactions plus m x n qPCR controls. When using ValidPrime® only m + n + 1 controls are needed. ValidPrime® will minimize the amount of control reactions and hence your costs as well as your efforts.

FEATURES

Offers a cost-efficient alternative to RT(-) controls Accurately validates qPCR assays in terms of their sensitivity to gDNA Allows correction for gDNA-derived signals

Reduces the need for DNase treatment USE VALIDPRIME® as a control for gDNA background in qPCR for normalization of samples to cell copy number as an endogenous control for CNV applications ValidPrime® is an assay to test for the presence of gDNA in test samples and when combined with a gDNA control sample, replaces all RT(-) controls.

ValidPrime® is highly optimised and specific to a non-transcribed locus of gDNA that is present in exactly one copy per haploid normal genome. The sequence detected by the ValidPrime® assay has no transcriptional activity and is not present in pure cDNA preparations. Therefore, ValidPrime® measures the number of genomic copies present in a sample. In an expression profiling experiment the ValidPrime® assay is added to the list of assays and the gDNA control is added to the list of samples. From the combined measurements of the ValidPrime® assay and the gene of interest (GOI) assays on all samples and on the gDNA control, the genomic background contribution to all RT-qPCR measurements can be assessed. ValidPrime® replaces the need to perform RT(-) controls for all reactions and makes RT-qPCR profiling easier and substantially cheaper.

The assay has very high PCR efficiency (E > 90% in tested commercial master mixes) and produces negligible amount of primer dimer products. Limit of detection (LOD) is estimated to 4 copies of DNA (0.01 ng of DNA) and limit of quantification (LOQ) is estimated to 32 copies of DNA (0.08 ng of DNA).

For ValidPrime® Human, ValidPrime® Mouse, and ValidPrime® Rat, a gDNA standard is provided which can be used to test the sensitivity of qPCR assays for gDNA background. ValidPrime® Vertebrate does not include a gDNA standard, since the standard needs to be specific for the species analysed and arranged by the user.

Overview

•     Detects contaminating gDNA in RNA preparations
•     No reverse transcription or external spike-ins required
•     Validated for human, mouse, and rat samples
•     Improves RT-qPCR specificity and result interpretation
•     Essential for RNA QC in diagnostics and biomarker studies

User Manual

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