A Step-by-Step Guide to Designing qPCR Primers

on 6th of February, 2013 in PCR & Real-time PCR
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About the Author:
I finished my Chemistry degree in 2007 at the University of Barcelona, Spain. From 2007-2009 I carried out a Master program in Biochemistry at the University of Barcelona, Spain. Since March 2009 I joined the Freiburg Institute for Advanced Studies (FRIAS) at the Albert-Ludwigs University Freiburg as a PhD student in the field of Systems Biology.

Primer design is a very important step while setting up your qPCR assay. If your primers anneal poorly or to more than one sequence, this can significantly impact the quality and reliability of your results. The good news is that primers are cheap, so you can test several different pairs and choose the best pair to use in your experiments. 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 primers for qPCR. 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 can see on the right corner of the screen a link to “Pick primers”.

Primer Parameters

PCR product/amplicon size: For efficient amplification in real-time RT-PCR, primers should be designed so that the size of the amplicon is <200 bp.

Number of primers to return: Up to you, but 10 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 or probes so that 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: Leave the default settings. The program will use then the Refseq mRNA sequence from the organism that you selected in the screen before to calculate the primers.

Checking the output screen

The primers should end with a C or G residue, because T and A residues can bind more easily to DNA in a non-specific way.

Optimal primers also have a GC content of around 50-60% to ensure maximum product stability.

Regarding self complementarity, the lower the better, to decrease the possibility of primer-dimer formation. Ideally the primer will have a near random mix of nucleotides.

Now you can pick the best two or three primers and test them. Good luck!

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4 thoughts on “A Step-by-Step Guide to Designing qPCR Primers”

  1. Avatar of evszany evszany says:

    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

  2. Avatar of saurav saha saurav saha says:

    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?

  3. Avatar of user-48565 user-48565 says:

    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.

  4. 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.

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