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Quantitative PCR (qPCR) primer design is a critical step when setting up your qPCR or reverse transcription-qPCR assay (RT-qPCR) for gene expression analysis. qPCR primers that anneal poorly or that anneal 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 reverse transcription PCR (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-quality 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 qPCR primer design 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.

qPCR Primer Design: Getting Started

The first step in designing primers is to get the nucleotide sequence of your gene of interest.

Go to the Pubmed gene database and search for your gene of interest. You can then filter by species in the right-hand corner of the next 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”). Note that there may be multiple sequences if your gene has different isoforms – make sure you click on the isoform you are interested in.

Click on the name and on the next screen, you will see a link to “Pick primers” in the right-hand corner of the screen underneath “Analyze this sequence”. Click on this link to take you to the Primer-BLAST tool.

Primer-Blast Tool

The Primer-BLAST tool has many parameters and options to set. The next sections take you through each of the options in the tool and explains what to set for each one and why.

Parameters for qPCR Primers

This section covers some of the basic settings for your primers, including the PCR product size and melting temperature. There is also an option to include sequences for reverse or forward 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 10 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 primer 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.

Exon/Intron Selection

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

Once all the parameters are set, clicking on ‘Get primers’ returns a list of potential forward and reverse primer sequences to choose from. This can take a while but the screen updates periodically to show the time since submission.

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, order and test them. Good luck!

If you have any other top tips for qPCR primer design, we’d love to hear from you in the comments!

Need help in other areas of qPCR from setup to data analysis? Check out our top 11 qPCR papers every researcher should know.

Need help making your PCR fail (a bit) less often? Download our free notorious PCR inhibitors poster and pin it up near your DNA engine. Or download the Bitesize Bio PCR eBook for more comprehensive practical guidance.

Originally published February 6, 2013. Reviewed and republished 2017 and April 2021.

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11 Comments

    1. Hey, you just open the FASTA sequence of the selected gene and on that page there is option to pick primer.

  1. What about with species without information at NCBI Reference Sequence?

    For example I work with Eucalyptus globulus and I can not use the tool “Pich primers” at NCBI.

  2. What about secondary structures?
    I also use primer-blast (nice indeed) or just primer3 and always check primers for hairpins and dimers (NetPrimer). Sometimes it’s turning out that primers “good” for primer-blast create plenty of potential secondary structures (?!).

    How about setting Mg, Na, primers concentrations in calculation? Does anybody use that figures? (I do).

    Of course in silico calculations aren’t perfect so it is crucial to keep track what primers work for particular instrument/setup, so next design could include real-life data.

    It is also true for determining Tm, as different settings and software gives very different results.

  3. I AM DESIGNING SOME DEGENERATE PRIMER IN MY PH.D WORK SO PLEASE TELL ME HOW TO DESIGN THE DEGENERATE PRIMER? AND ALSO TELL ME THAT THE DESIGNING OF DEGENERATE PRIMER SHOULD BE THE SAME SPECIES OR TOOK FROM ANOTHER SPECIES?

  4. I have been using the method you suggested and it has been working well. However, I have run into a problem. On one gene the I have received “Products on potentially unintended templates” on every primer that has come out. Are these a problem when they show up? Are there any suggestions you can give me as to messing with the settings to avoid these?

    Thanks

    1. I have the same problem. I have been trying to design primers and 50% of time, on each gene, all the primers would give unintended templates, as there are some variants! I am stuck!

      Thanks!

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