“Based on the qPCR data I have seen in our lab and from researchers looking for support, I learned to appreciate the value of fully validated assays,” said Dr. Jan Hellemans, co-CEO of Biogazelle, a qPCR data analysis and services company he cofounded with Prof. Jo Vandesompele in 2007 as a privately held Ghent University spin-off. Hellemans and Vandesompele were also coauthors of the MIQE guidelines (minimum information for publication of quantitative real-time PCR experiments) established to promote high-quality data from qPCR experiments (Bustin et al. 2009).
Commercial providers have traditionally used software to facilitate the design of qPCR assays. While these algorithms detect sequences such as single-nucleotide polymorphisms and secondary structure that may impair or bias amplification, they do not guarantee that the assay will work in the lab. To validate an assay, users must still test the resulting PCR primers for efficiency and specificity with the target sequence.
Meeting the MIQE Guidelines through Full Wet-Lab Validation
Bio-Rad collaborated with Biogazelle to design, optimize, and experimentally validate SYBR® Green-based gene expression assays for the mouse genome. PCR primer pairs for each mouse transcript were designed following strict guidelines on maximum transcript coverage, minimal overlap with known SNPs, and spanning large introns where possible.
In accordance with the MIQE guidelines, every assay was wet-lab validated following strict assay performance standards for amplification efficiency, specificity, sensitivity, and linear dynamic range. Assay specificity was confirmed using next-generation sequencing (NGS). This approach yielded validated assays for more than 90 percent of the mRNA transcriptome.
“Using Bio-Rad’s PrimePCR assays now allows users to run high-quality MIQE-compliant assays without having to spend the effort performing the lab validation themselves,” said Hellemans.
Additionally, the PrimePCR mouse assays are:
• Expertly curated — predesigned pathway and disease plates curated by Thomson Reuters for canonical pathways and related collections help researchers identify and investigate key targets
• Customizable — easy-to-use plate configurator allows users to lay out assays on a plate exactly as they choose or use a suggested plate layout template
• Fully integrated — full integration with CFX real-time PCR systems and data analysis software streamlines data collection and analysis