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

• RTPrimerDB is an excellent database storing known primer and probe sequences for popular techniques (SYBR Green I, Taqman, Hybridisation Probes, Molecular Beacon). This can help you save the time of designing and testing your own primers. It is also intended to facilitate standardisation among different laboratories. The database is hosted by the Center for Medical Genetics, Gent, Belgium. Please submit you tested primer pairs.

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

• PrimerBank. From the website "PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). PrimerBank contains over 306,800 primers covering most known human and mouse genes. ...The primer design algorithm has been extensively tested by real-time PCR experiments for PCR specificity and efficiency. We have tested 26,855 primer pairs that correspond to 27,681 mouse genes by Real Time PCR followed by agarose gel electrophoresis and sequencing of the PCR products. The design success rate is 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. Link to paper.

• qPrimerDepot. From the website "This database provides qRT PCR primers for 99.96% 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 approximately 60C". Link to paper.

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

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 biger 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