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A Step-by-Step Guide to Designing qPCR Primers

qPCR primers

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.

Getting Started

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.

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

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.

qPCR primers
Image Credit: Ulf Liljankoski

11 Comments

  1. molbiologist on April 11, 2019 at 9:44 pm

    It is crucial to reflect monovalent cation, Mg2+, and dNTPs concentrattion in the Tm calculation and to check the potential primer dimers. A suitable online application is Primer Inspector at http://www.molbiotools.com/primerinspector.php

  2. Isc on July 20, 2016 at 6:04 pm

    When I try to use the method you suggested, it tells me that my template is too long, and to select a ‘from’ for my forward primer and a ‘to’ for my reverse. Isn’t the whole point of the program to find a good ‘from’ and ‘to’ point, in order to match my requirements (PCR product size, spaniing an exon-exon junction, etc)

    I’ve limited my pcr product size to 100-200bp, so I don’t quite see how the template is too large. Am I missing something?

  3. HongNguyen on June 20, 2016 at 9:20 am

    I would like to design random primers which can amplified just whole genome of bacteria, what could I do? Could you help me, please!

  4. i on February 10, 2016 at 10:08 pm

    hye!
    i cannot find the link to “pick primer”.
    help me please.

    • Sulekha on November 5, 2016 at 9:54 am

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

  5. Rodrigo Hasbun on March 26, 2014 at 4:37 pm

    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.

    • Abdullah on December 13, 2016 at 7:42 pm

      Copy the gene sequence into ncbi-primer-blast and go from there

  6. user-48565 on November 19, 2013 at 1:17 am

    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.

  7. saurav saha on September 26, 2013 at 12:41 pm

    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?

  8. evszany on April 16, 2013 at 5:23 pm

    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

    • Mary on June 27, 2019 at 4:11 pm

      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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