Control for genomic background
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.
- 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
- 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.