How qPCR primers differ from PCR primers
A qPCR measures how quickly a product builds up, cycle by cycle, so the primers have to copy their target at close to 100% efficiency and make one product only. That calls for short amplicons and tighter rules than a cloning PCR, where any amount of the right band will do. The general rules are in How to Design PCR Primers; these are the ones that change for qPCR.
The rules
- Amplicon: about 70 to 150 bp. A short product is copied completely in every cycle, which keeps the efficiency high.
- Primer length: 18 to 25 bases.
- Tm: about 58 to 62 °C, with the two primers within 1 to 2 °C of each other, worked out for the salt and magnesium of your master mix.
- GC content: 40% to 60%, with no more than 3 G or C in the last 5 bases at the 3′ end.
- Avoid: runs of 4 or more of one base, especially G, and stretches that pair with the primer itself or with the other primer. Primer dimers matter most with SYBR Green, which lights up any double-stranded DNA, dimers included.
Probes
A hydrolysis (TaqMan) probe binds between the two primers and is designed with a Tm about 5 to 10 °C above theirs, so it is already bound when the primers start to extend. Avoid a G at its 5′ end, next to the reporter dye, because a G there quenches the fluorescence.
Measuring mRNA: span an exon junction
For gene expression from cDNA, place one primer across an exon–exon junction, or put the two primers in different exons with a long intron between them. Genomic DNA left in the RNA then gives no product, or one too long to amplify well. A no-RT control, RNA that went through the reaction without reverse transcriptase, shows whether any genomic DNA still gets through.
Check the primers before ordering
- Calculate both Tm values with the Primer Tm Calculator, using the magnesium of your master mix, which is often 3 mM or more.
- Look for hairpins, self-dimers and a weak or sticky 3′ end with PCR Primer Stats.
- Paste the template into PCR Products to confirm a single product of the expected size, and search the primers against the whole genome, for example with NCBI Primer-BLAST, to rule out other targets.
Check them in the lab
Run a dilution series of template, five or more 10-fold steps, and plot Ct against the log of the amount with the Standard Curve Calculator. The slope gives the efficiency: E = 10−1/slope − 1. A slope of −3.32 means 100%, and 90% to 110% is generally accepted. With SYBR Green, a melt curve with one sharp peak shows a single product, and the no-template control should stay flat. Then work out relative expression with the ΔΔCt Calculator, which uses the Pfaffl method when the two primer pairs' efficiencies differ.