“PCR primer design and assay validation are two of the most important steps for ensuring accurate, reproducible, and consistent quantitative PCR results,” said Jo Vandesompele, an author of the MIQE research paper. “Unfortunately, not all qPCR users are aware that qPCR assays in the industry have not always been fully validated.”
Other companies offering “validated” primer assays simply validate their primer design algorithm and do not wet-lab test each assay to ensure that they all meet MIQE guidelines. While all commercial assay providers use computer-based algorithms to design primers for qPCR, this alone does not guarantee that the assay will actually work in the lab. Thus, to ensure MIQE compliance, researchers still have to wet-lab validate their assays, which can waste time and resources.
“If a vendor claims to have wet-tested its primers, ask two questions to ensure your real-time PCR experiment will succeed,” said Sam Ropp, Marketing Manager of Bio-Rad’s Gene Expression Division. “One, did they actually test your primer pair, and two, was it up to MIQE standards? Testing amplification efficiency without a serial dilution won’t cut it.”
Meeting the MIQE Guidelines through Full Wet-Lab Validation
Bio-Rad collaborated with Jo Vandesompele and Jan Hellemans of Biogazelle, a qPCR data analysis and services company, to design, optimize, and experimentally validate every primer pair for the protein coding genes that make up the human transcriptome. All assays were designed following strict guidelines on maximum transcript coverage, minimal overlap with known SNPs, and spanning large introns where possible.
In addition, Bio-Rad validated every assay in the lab. Bio-Rad provides full transparency on the performance of each of its PrimePCR assays in the form of a standardized assay validation report. In accordance with MIQE guidelines, assays were rigorously evaluated for:
• Efficiency — calculated from the results of a tenfold dilution series of synthetic templates; only assays that displayed good linear performance and efficiency were judged to be of sufficient quality
• Specificity — all PCR products from cDNA were validated by massively parallel sequencing, which overcomes the limitations of melt curve analysis and gel electrophoresis
• Sensitivity — nonspecific amplification was minimized by redesigning assays with unacceptably high background signal (mainly primer dimers)
PrimePCR products are available as individual SYBR® Green assays, preplated pathway and disease-specific panels, or custom configured plates. The panels were designed from canonical pathway maps provided via the systems biology solutions from the Life Sciences team at Thomson Reuters. The pathway curation and ranking strategy employed by Thomson Reuters and Bio-Rad ensures that the gene assays present on each real-time PCR pathway and collection plate are the most relevant for gene expression profiling based on differential expression studies and the frequency with which gene targets appear in the peer-reviewed literature.
To further support qPCR researchers, PrimePCR assays and panels are fully integrated with Bio-Rad’s CFX real-time PCR systems and CFX Manager™ 3.0 data analysis software for streamlined data collection and analysis.
For validation reports and more information on assay design and validation, please visit www.bio-rad.com/PrimePCR