I've been trying to design some qPCR primers lately. I am aware that the ideal primer should span an exon-exon junction. However, two of my gene of interest (GPR109a and GPR40) have only one exon. In this case, what is the best approach to select primers with the best chance of rightly amplifying my target genes?
The parameters will be the same as any other primer:
- amplicon
The parameters will be the same as any other primer:
- amplicon
Dear Babajide
I cannot really add to Andres excellent answer; In fact he anticipated my own point of making sure that, if you design primers internally within exons make sure that your genomic DNA is digested away thoroughly so that competing reactions within genomic DNA itself do not occur to any substantial degree which reduces priming efficiency associated with cDNA; which is paramount in qPCR (must be > 85% and certainly no lower than 75% and that being so you MUST ensure that the difference in priming efficiency between your GOI and housekeeper is no more than 5% and that you use the Pfaffl method of gene expression analysis instead of the more common Livak or delta delta ct analysis, which pre supposes an approximate doubling of product during PCR; But that is digressing)
Let me add a couple more things to expand on Andres excellent points:
1. When you say you only have one exon do you mean one coding exon, i.e. the CDS is in fact just one exon. That being so there will be an upstream 5' UTR and downstream 3' UTR in the non coding RNA also present in the cDNA made by reverse transcription. In fact many groups deliberately situate primers at the coding exon 3' UTR boundary
2. With reference to situating primers within exons and therefore ensuring all traces of gDNA are eradicated, a DNASE I like Turbo from PE (ambion) is infact and rDNAse, devoid of contaminating RNAse and therefore able to aggressively breakdown to completion gDNA @ 37C for 1 hour without at the same time breakdown the parent RNA, inhibiting efficient RT:
https://www.thermofisher.com/order/catalog/product/AM2238
In contrast most purified DNases carry a minute fraction of RNase and therefore cannot be used to digest RNA @ 37C for long periods (> 15 min) without incurring simultaneous breakdown of RNA which subsequently undermines efficient RT
3. Keep qPCR amplicons @ 100-200bp in order to maximise the efficiency of primers
4. Expanding on general primer design from the above find attached an SOP detailing primer design and screening algorithms
Hope this helps !
Two great answers above. The only thing I'd add is to take the time to ensure that your reactions run to close-to-equal efficiencies and you can interrogate your RNAprep with a PCR for a promoter region (we use CD4 for example) to satisfy yourself that it meets your criteria for DNA-free, prior to the RT step.
Good Luck!
G
Nice addition Grant
with regard to primer efficiencies I try and ensure that the GOI and housekeeping ref primer are within 5% of one another
Grant suggestion is very good. I never considered that. We always learn at Research Gate.
Thank you all so much for the enlightening responses. We always run DNase step irrespective of primer design. However, I'll definitely look into running a no RT-control while ensuring adequate reaction conditions as suggested. The possibility of interrogating the promoter region is very interesting. Again, I'm very grateful for the prompt and invaluable responses.
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