PCR Primers, Probes, and Panels

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PCR Primers for Gene Expression Assays

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Design and Validation of Real-Time PCR Primers

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

Sensitivity Accurate detection of 20 copies
Specificity Amplicon sequence validated with next generation sequencing (NGS). Minimal primer dimer formation and genomic DNA cross reactivity.
Amplification Efficiency 90–110%
Linear Dynamic Range Minimum of six orders of magnitude. Detection of a synthetic template standard curve from 20 to 20 million copies.
R2 >0.99

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

Pathway Curation

Thomson Reuters provided interactive pathway maps for 260 canonical pathways. Each pathway belongs to one or more general biological categories such as cancer. The pathway maps illustrate protein interactions and regulation to provide a comprehensive picture of signaling and disease processes. The selected pathways were used to design panels of real-time PCR primers tailored for the top-ranked genes for differential gene expression analysis. Each gene target within a pathway was assigned a score based on the frequency of differential expression and its research significance. The resulting scores were used to select the assays included in the corresponding real-time PCR pathway panel.

PrimePCR Pathway Analysis: Pathway Curation and Real-Time PCR Panel Design Strategy

Control and Reference Assays for Real-Time PCR

Control assays and synthetic DNA templates were designed to facilitate the assessment of the key experimental factors impacting your real-time PCR results.

DNA Contamination Control Assay
Use the PrimePCR DNA contamination control assay to determine if genomic DNA (gDNA) is present in a sample at a level that may affect PCR results. This assay may also be used to compare relative levels of gDNA contamination present in different samples to determine if PCR results may be affected.

Positive PCR Control Assay
Use the PrimePCR positive control assay to qualitatively assess the performance of a PCR reaction associated with a single sample. This assay may also be used to compare the relative performance of PCR reactions associated with different samples.

RNA Quality Assay
Use the PrimePCR RNA quality assay to determine if RNA integrity may adversely affect PCR results for a single sample. This assay may also be used to compare relative RNA integrity among different samples to determine how PCR results might be affected.

Reverse Transcription Control Assay
Use the PrimePCR reverse transcription control assay to qualitatively assess the performance of the reverse transcription reaction associated with a single sample. This assay may also be used to compare the relative performance of the reverse transcription reactions associated with different samples.

Reference Gene Assays
Reference genes are used in relative gene expression analysis to normalize for variation in the amount of input messenger RNA (mRNA) among samples. To ensure accurate quantitation, it is important to include one or more reference genes exhibiting constant expression levels under the experimental conditions. To streamline reference gene selection, we offer PCR primers for a set of commonly used reference genes that can be used individually, easily screened using our preplated 96-well and 384-well reference panels or added to custom-designed plates.

Show Reference Gene Assays
View Reference Gene Assay Panels


Real-time PCR primer assays consist of unlabeled PCR primer pairs for use with dye-based chemistry such as SYBR® Green or EvaGreen®. Probe assays for real-time PCR and Droplet Digital™ PCR include PCR primers and a dual-labeled fluorescent probe with your choice of fluorophore. You can also design custom PCR primer or probe assays.

Find an Assay or Template

Select an individual PCR primer or probe assay for PreAmp, qPCR, or ddPCR.

Find a Pathway Plate

Find a predesigned PCR plate for your biological pathway or disease of interest.

Create a Custom PCR Plate

Design a custom 96-well or 384-well PCR plate.

PrimePCR Assay Selection Guide

 

Real-Time PCR Assays

Droplet Digital PCR Assays

Organisms Human, mouse, rat
Human
Assay Chemistry Dye-based and probe assays Probe assays
Applications Gene expression
Preamplification
Copy number variation
Mutation detection
Formats Individual assays
96- and 384-well predesigned plates*
96- and 384-well custom plates*
Individual assays

* Probe assays are not available in plates

PCR Primer and Probe Assays for Real-Time PCR

Our real-time PCR primers were designed in collaboration with leading experts in real-time PCR research. Every PCR primer pair has been experimentally validated to ensure optimal assay performance. For more information on the validation of the DNA primer pairs, see Bulletin 6262, PrimePCR Assay Validation.

Our fluorescently labeled oligonucleotide probes were designed to enable multiplexing. These 5' nuclease probes were designed for maximum specificity and transcript coverage. qPCR probe assays are available with a FAM, HEX, TEX615, Cy5, or Cy5.5 fluorophore.

Real-time PCR assays are available in the following formats:

  • Predesigned primer and probe assays — gene expression assays for the human and mouse genomes
  • Custom oligonucleotides — design your own PCR primer or probe assay
  • Customizable 96- and 384-well platesuse the custom plate configurator to conveniently design a PCR plate
  • Predesigned pathway panels — select from a wide variety of disease- and pathway-specific panels
  • PreAmp assays — 1,000-fold unbiased target-specific preamplification of limited nucleic acid

Controls and Reference Assays are available to facilitate assay normalization and control:

  • Experimental Controls — for evaluating reverse transcription performance, RNA quality, genomic DNA contamination, and PCR reaction performance
  • Reference Gene Assays — for relative gene expression analysis to normalize for variation in the amount of input messenger RNA (mRNA) between samples
  • DNA templates — serve as a positive real-time PCR control for the corresponding gene assay

All real-time PCR assays and panels can be used with CFX real-time PCR detection systems and data analysis software, streamlining PCR data collection and analysis.

Probe Assays for Droplet Digital PCR

Droplet Digital PCR (ddPCR™) technology provides an absolute measure of target DNA molecules with unrivaled accuracy, precision, and sensitivity, accelerating discovery and enabling new research strategies for inherited disorders, cancer, and infectious disease. Probe assays for the QX200™ Droplet Digital PCR system enable the fine quantitative discrimination required to resolve small fold changes without a standard curve. ddPCR probe assays are available with a FAM or HEX fluorophore.

For more information, see Bulletin 6512.

ddPCR assays are available in the following formats:

  • Predesigned mutation detection probe assays — for human samples
  • Predesigned copy number variation (CNV) probe assays — for human samples
  • Custom oligonucleotides — design your own ddPCR probe assay

PrimePCR assays, plates, and controls have a guaranteed shelf life of 12 months when stored at 4°C.

PrimePCR™ Analysis Software

Download GeneStudy_1.0.030.1023, our custom data analysis software that enables gene expression analysis of PrimePCR data generated on any real-time PCR instrument. Analysis options include bar chart, clustergram, scatter plot, volcano plot, and heat map analysis.
Number Description Download
LIT10026370 Instruction Manual, PrimePCR Assays, Panels, and Controls, Rev E Click to download
10039761 Instruction Manual, PrimePCR Quick Guide, Rev A Click to download
10042030 PrimePCR™ PreAmp Assay Quick Guide, Rev A Click to download
10047489 PrimePCR™ ddPCR™ Mutation Detection Assays (in silico designed) Product Insert, Rev A Click to download
6262 PrimePCR Assays: Meeting the MIQE Guidelines by Full Wet-Lab Validation, Rev B Click to download
6263 PrimePCR Pathway Analysis: Pathway Curation and Real-Time PCR Panel Design Strategy, Rev B Click to download
6290 PrimePCR Assays and Panels Brochure, Rev D Click to download
10033487 PrimePCR ddPCR Mutation Detection Assays Product Insert, Rev B Click to download
10033173 PrimePCR ddPCR Copy Number Variation (CNV) Assays Product Insert, Rev B Click to download
10041702 PrimePCR ddPCR Gene Expression Assays (EvaGreen) Product Insert, Rev A Click to download
6512 PrimePCR Assays for Droplet Digital PCR Brochure. Rev A Click to download