Choosing primers for qPCR

What are the best primers for my qPCR experiment? Choosing suitable primers is an early crucial step in your quantitative reverse transcriptase PCR experiment - QRT-PCR. Reusing a tested primer pair from a repository or publication can save you some time. Otherwise primer selection from scratch is similar to that for a standard qualitative PCR experiment with some small variations.

Primer repositories and collections

Primer databases can help you save the time of designing and testing your own primers. It is also intended to facilitate standardisation among different laboratories.

• A gene- or transcript-specific primer database for quantitative real-time PCR This user-friendly plateform uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally demonstrating their high quality and demonstrating high transcript specificity in complex samples. Until now, you could retrieve primers in a high-throughput fashion for all Homo sapiens, Mus musculus, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio genes in assembled chromosomes annotated in the Ensembl database. Publication describing the database: [1]

• Quantitative PCR Primer Database (QPPD) provides information about primers and probes that can be used to quantitate human and mouse mRNA by reverse transcription polymerase chain reaction (RT–PCR) assays. All data has been gathered from published articles, cited in PubMed. This resource is a collaborative effort of the NCI-Frederick Gene Expression Laboratory and the CGAP Genome Annotation Initiative. In association with the Center for Cancer Research and the Laboratory of Population Genetics.

• RTPrimerDB is a collection of user submitted qPCR primers and probe sequences mostly human (2835), mouse (624), and rat (388) based on stats from 2/08. The database includes SYBR Green I, Taqman, Hybridisation Probes, and Molecular Beacon. Other features include: mfold secondary structure prediction, primer alignment, and BLAST. The database is hosted by the Center for Medical Genetics, Gent, Belgium. Please submit you tested primer pairs. Publication describing the database [PMID 16381959].

• PrimerBank is a public database for human and mouse qRT-PCR primers. PrimerBank contains over 306,800 primers covering most (percentage??) known human and mouse genes. The creators of the repository report that 26,855 primer pairs tested corresponding to 27,681 mouse genes had a design success rate of 82.6% (22,187 successful primer pairs) based on agarose gel electrophoresis. Don't neglect to check the efficiency and specificity of the oligos yourself though. Primers are designed to have a Tm of 60-63°C and a product of 100-250nt [2]. Link to paper.

• qPrimerDepot is a public database for human qRT-PCR primers searchable with either RefSeq ID or HUGO gene name. It claims to contain 99% of human RefSeq sequences. For 99% of intron-bearing genes, the PCR product will cross an exon-exon border which overlaps one of the largest introns. All primers have annealing temperatures of approximately 60°C. Link to paper.

• Check out the Eccles Lab collection of human and mouse qPCR reference genes on OWW.

• RealTimePrimers.org is another such database but it contains only few primers.

Primer design

An excellent and fast way to select primers is with the free online-tool Primer3, currently in v0.3. Primer3Plus, a variation of Primer3 has qPCR settings. Or just apply the following or similar settings to Primer3:

• pair towards 3' end (often more specific, some cDNAs don't contain)
• pair separated by an exon-exon boundary (reduces genomic background) e.g. last exon & penultimate
• amplified region must be no bigger than 200 bp; usally 60-150 bp
• GC content: 50-60%
• min length: 18, max length 24 (best: 20 nt)
• melting temperature: min 60, max 63, best 60
• max Tm difference: 10 (shouldn't be more than 1 in final pair)
• max 3' self complementary: 1
• max poly-x: 3

Verify by blasting the primers sequences. Target gene should come out with the lowest E value. No other gene should be close. Also check whether possible isoforms will be detected by the candidate primer pair. See also: Designing primers

Common errors

• primer not gene specific (blast to check)
• primer form dimers or secondary structure (use prediction software to discard those primers)
• primer in region of SNP - this may changes amplification efficiency between different individuals

External links

• Primer and Probe Design for Quantitative Assays from Uni of Illinois Chicago
• GeneScript TaqMan probe design tool
• TaqMan design tips using Primer Express, Yale (note Primer Express is commercial software single license <8000€)

• more links from protocol-online.org
• qPCR link list from Health Science Library, Uni Pittsburgh