Bioradiations Staff
Last year Bio-Rad introduced new expertly designed and experimentally validated assays for real-time PCR — the PrimePCR assays and panels. PrimePCR assays achieved a new level of performance and validation utilizing SYBR® Green detection chemistry for optimal gene expression analysis. Now the PrimePCR portfolio is greatly expanded to include new chemistries, technologies, applications, and species.
The expanded PrimePCR product portfolio now includes:
- Individual SYBR® Green assays covering the human and mouse genomes
- Individual qPCR probe assays available for human and mouse targets
- Individual ddPCR™ probe assays for copy number variation (CNV) and mutation detection
- Predesigned pathway and disease panels
- Custom configured PCR plates
- Custom SYBR® Green and probe assays
- Control SYBR® Green and probe assays
- Reference gene assays and panels
Enhanced Web Ordering Tools
Bio-Rad’s new online ordering PrimePCR website allows researchers to quickly choose the technology (qPCR or ddPCR), chemistry (SYBR® Green or probe), application (gene expression, copy number, or mutation detection), and organism (human or mouse) of interest (see Figure 1). The repertoire of expertly designed plates can be searched or sorted to discover the best match for a predesigned panel for pathway or disease analysis of interest.
Fig. 1. Screen shot of online filtering tool.
Mouse Genome
Assay Performance Standards and Efficiencies
| Sensitivity | Accurate detection of 20 copies |
| Specificity | Validated amplicon sequence with next generation sequencing (NGS). Minimal primer dimer formation and genomic DNA cross reactivity |
| Amplification efficiency | 90–110% |
| Dynamic linear range | Minimum of six orders of magnitude. Detection of a symbolic template standard curve from 20 to 20 million copies |
| R² | >0.98 |
Fig. 2. Validation criteria table for SYBR Green mouse assays
Bio-Rad recently launched PrimePCR™ SYBR® Green assays and panels for the mouse genome. Similar to our original offering of gene expression assays for the human genome, every assay in the mouse genome has been wet-lab validated and meets the stringent performance criteria detailed below (see Figure 2). By expanding the species offerings of PrimePCR assays, we enable more researchers to save time by eliminating in-house validation and optimization effort, while increasing confidence in their qPCR data.
Real-Time PCR Probe Assays
The probe assays for qPCR gene expression analysis have been developed in partnership with Biogazelle, leaders in real-time PCR. Each assay is available labeled with a FAM, HEX, or Tex 615 fluorophore and has a gene-specific synthetic template designed for a positive real-time PCR result. Bio-Rad offers a complete workflow for real-time PCR including RNA isolation kits, reverse transcription kits, real-time PCR supermix, real-time PCR instruments, and data analysis software. Probe assays are recommended to be used with the iScript Advanced cDNA synthesis kit for RT-qPCR and SsoAdvanced Universal Probes supermix for guaranteed performance.
Digital PCR Probe Assays
Droplet Digital PCR (ddPCR™) technology (described in detail in the Applications & Technologies section of the Bio-Rad website) provides an absolute measure of target DNA molecules with unrivaled accuracy, precision, and sensitivity. Droplet Digital PCR probe assays for the QX100™ Droplet Digital PCR system and QX200™ Droplet Digital PCR system (launching soon) provide single-copy PCR resolution without a standard curve. ddPCR probe assays can accelerate discovery and enable new research strategies for inherited disorders, cancer, and infectious disease. ddPCR probe assays are available with a FAM or HEX fluorophore and can be used for copy number and mutation detection applications. Bio-Rad’s initial launch of digital PCR assays includes 66 high-impact targets, with more on the horizon. All ddPCR probe assays have been experimentally tested to meet performance requirements. Validation reports are available online and are included with every assay. Examples of validation data for CNV assays (Figure 3) and mutation assay (Figure 4) are shown below.
PrimePCR™ ddPCR™ CNV Assay Validation Data
Examples of human BRCA1 CNV assay validation data are shown below.
Fig. 3. 2-D amplitude scatter plot. Single-well data of target assay (FAM, Channel 1) duplexed to EIF2C1 reference assay (HEX, Channel 2).
Fig. 4. CNV sample replicate plot. Copy number analysis of two samples, using the EIF2C1 reference assay. Technical replicates are shown.
PrimePCR ddPCR Mutation Assay Validation Data
Examples of human BRAF, p.V600E mutation assay validation data are shown below.
Fig. 5. Fractional abundance (%). Dilution series of mutant DNA in ~40,000 copies of wild-type DNA background.
Fig. 6. ddPCR amplitude scatter plot (wild type + mutant). Single-well data for mutant DNA spiked into wild-type DNA (~0.1%). Mutation assay (FAM, Channel 1) was duplexed with the corresponding wild-type reference assay (HEX, Channel 2).
Fig. 7. ddPCR amplitude scatter plot (wild type). Single-well data for wild-type DNA (no mutant). Mutation assay (FAM, Channel 1) was duplexed with the corresponding wild-type reference assay (HEX, Channel 2).
SYBR is a trademark of Life Technologies Corp. Bio-Rad Laboratories, Inc. is licensed by Life Technologies Corporation to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes only.