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 <200 bp.
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).
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!
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