qPCR primers

A Step-by-Step Guide to Designing qPCR Primers

Primer design is a very important step when setting up your qPCR or reverse transcription-qPCR assay (RT-qPCR). If your qPCR primers anneal poorly or to more than one sequence during amplification, it 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. A poor primer will result in both inefficient reverse transcription and inefficient amplification – a lose-lose situation. So you better take your time when picking your primers!

The good news is that primers are cheap, so you can 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. I like to use the NCBI tool Primer BLAST to design qPCR primers.

Here are the main steps to design primers using this free program:

Go to the Pubmed gene database and search for your gene of interest. You can filter by species in the right corner of the screen. Click on the gene of interest and scroll down until you find the NCBI Reference Sequence (RefSeq) of 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 size of the amplicon is
  • Number of primers to return: This is up to you, depending on how many you want to look at. Ten won’t take too long to calculate and will give you plenty of options to choose from.
  • Melting temperature: As a rule, aim for a minimum of 57°C and a maximum of 63°C; the ideal melting temperature is 60°C (with a maximum difference of 3°C in the Tm’s of the two primers).

Exon/intron Selection

To avoid amplification of contaminating genomic DNA, design primers so one half 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 calculate 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 50-60% 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!


  1. 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?

  2. 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!

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

    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.

  4. 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

  5. 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.

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


  7. 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?


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