Dear everyone,
May I please get your help with primer dimer issue in sybr green qPCR reaction?
Our lab is working on developing diagnostic methods based on cell-free RNA from plasma. We are trying out several cell-free RNA extraction kits.
To test the product, we designed primers to amplify housekeeping genes such as actin and GAPDH in both PCR and qPCR reactions. After trying out several pairs of primers, we found out that only one among 3 pairs of actin primer is usable. It resulted in decent amplification products with good melt curve in sybr green based qPCR reactions. The other two pairs have very variable melt curve.
When using the good primer pair, we confirmed that in the qPCR reaction we do have amplification of actin. The confirmation is done via gel electrophoresis and sequencing of qPCR products.
Sadly, the Ct values of our negative (non-template) control is not very stable. Replicates in the same experiments have the same Ct values but the Ct values vary from experiments to experiments (21-31). Although there is still clear Ct separation between reactions with cDNA samples and the negative control (i.e. (fairly stable delta Ct)), this situation is not ideal. The primer dimer band also looks very obvious on the agarose gel. If we want to develop diagnostic kit, this is not acceptable. Do you have any advice on what we should do to solve this primer dimer issue and Ct-value variable issue?
We couldn't find any GAPDH primers that are specific for GAPDH mRNA. All primers have non-specific amplifications. Therefore we have not tried this out many times. Other house-keeping genes have not been reported to express in cell-free RNA plasma so we have not tried them.
Thank you very much for your time and advice!
Best,
Huong
Hello
If GAPDH mRNA can be found as cell-free RNA, I dont see any reason why other reference genes mRNA could not be found! Therefore I recommend if you cannot design a good primer for GAPDH, try for another reference gene and be the first one!
beta ACTIN primers might be a good option. It is a very reliable housekeeping gene.
-Avoid complementary clamps on the 3' end of your primers, e.g. GC or GCGC, -Measure and adjust your primer and template concentrations to recommended values.
-Avoid mixing primer pairs within the same tube.
Good luck!
Primer dimer can become a real headache that they could appear as a real amplification on agarose gel at times, i.e. it shows up as the desired molecular weight but not all the way at the lower end of the gel. I assume that the problematic Ct value is the qPCR version of the above stated.
In the past when I troubleshoot my agarose primer dimer problem (that was actually the mouse genotyping) I have tried cold-preparation-hot-start, touch-down, playing around with the primer/MgCl2 concentration, new reagents, new pipettor, another pair of hands, swearing and praying, and at the end of the day I solved it by screening many pairs of primers (designed by myself, the fancy term is "customized"). That wasn't too bad compared to the other things I tried. And it is actually worth noting that when you work with in vivo DNA/RNA/cDNA, it is not as straight forward as when working with PCR product or nucleic acid extracted from simpler/common in vitro cultures. Primers will prime to anything that it can bind to and render the previously working primers to be problematic. That's why diagnostic test kit development is not easy and needs some work around during the process.
May be you can spike with HIV derived virus like particle (VLP) and develop qRTPCR based assay for VLP. Use this for normalisation/quatitation from cell free assays.
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