Bio-Rad collaborated with Biogazelle, leaders in real-time PCR research, to design and experimentally validate PCR primers for gene expression assays across the human and mouse transcriptomes. All PCR primers were designed to meet stringent performance standards following the MIQE guidelines (minimum information for publication of quantitative real-time PCR experiments; Bustin et al. 2009).
Assay Performance Standards
||Accurate detection of 20 copies
||Amplicon sequence validated with next generation sequencing (NGS). Minimal primer dimer formation and genomic DNA cross reactivity.
|Linear Dynamic Range
||Minimum of six orders of magnitude. Detection of a synthetic template standard curve from 20 to 20 million copies.
These DNA primer pairs were designed by prioritizing the gene regions most commonly found in transcript variants. Strict design criteria were used to ensure optimal real-time PCR results for each target:
- Target regions without SNPs
- PCR primer pairs annealing across intron/exon junctions when possible
- No secondary structure in primer annealing sites
- Maximum number of transcript isoforms detected
- PCR primers compatible with standard assay conditions
Every PCR primer pair was experimentally validated using Bio-Rad’s iScript™ advanced cDNA synthesis kit and SsoAdvanced™ SYBR® Green supermix. PrimePCR assay design and validation are fully described in the following publication.
PrimePCR Assays: Meeting the MIQE Guidelines by Full Wet-lab Validation