Primer design is a critical step when setting up your qPCR or reverse transcription-qPCR assay (RT-qPCR). qPCR primers that anneal poorly or to more than one sequence during amplification can significantly impact the quality and reliability of your results. Also, if you are performing a one-step RT-qPCR, the reverse transcriptase will use the reverse primer to prime the transcription reaction. In this scenario, a poor primer would result in both inefficient reverse transcription and inefficient amplification – a lose-lose situation.
Considering the above, it’s well worth spending the time necessary to design good qPCR primers. This article will tell you exactly how to do that!
The good news is that primers are cheap, so you can easily test several different pairs to choose the best ones for your experiment. The bad news is that primer testing requires time and patience, so the sooner you get a pair of primers working, the better.
The NCBI tool Primer BLAST is widely used for qPCR primer design. There are many other primer design tools available online, including primer3, and PCR suppliers often offer their own design programs free of charge.
Below are the main steps involved in designing primers using the NCBI tool Primer BLAST. The design steps will be similar if you use other primer design programs, and the information below should give you an idea of the parameters to watch out for.
Go to the Pubmed gene database and search for your gene of interest. You can filter by species in the right-hand corner of the screen. Click on your gene of interest and scroll down until you find the NCBI Reference Sequence (RefSeq) for your gene (e.g., “NM_203483”). Click there and in the next screen you will see a link to “Pick primers” in the right corner of the screen.
Parameters for qPCR Primers
Set the following primer parameters:
- PCR product/amplicon size: For efficient amplification, design the primers so that the amplicon is between 70 and 200 bp long.
- Number of primers to return: This is up to you, depending on how many options you want to choose from. It won’t take long for the program to design ten primer pairs, and this should give you a reasonable chance of finding a suitable pair.
- Melting temperature: As a rule, aim for a minimum of 60°C and a maximum of 63°C; the ideal melting temperature is 60°C (with a maximum difference of 3°C in the melting temperatures, Tm, of the two primers). You can use a Tm calculator to determine these temperatures.
To avoid amplification of contaminating genomic DNA, design primers so that one half of the primer hybridizes to the 3′ end of one exon, and the other half to the 5′ end of the adjacent exon. To do this, simply select “Primer must span an exon-exon junction.” You don’t need to change the other settings.
Primer pair specificity checking parameters: Use the default settings. The program will use the RefSeq mRNA sequence from the organism you selected to design the primers.
Checking the Output Screen
Take a look at the options the program returned and pay special attention to the following:
- Make sure the 3′ end of the primer contains a C or G residue, because T and A residues bind more easily to DNA in a non-specific way.
- Aim for a GC content of around 40-60% to ensure maximum product stability.
- Avoid self complementarity to decrease the possibility of primer-dimer formation. Ideally, the primer should have a near random mix of nucleotides.
Now, pick the best two or three primers and test them. Good luck!
Originally published in 2013. Updated and republished in 2017.