I have run RT-PCR on 3 sets of HEK 293 samples and obtained divergent melt curves for one gene (Bovine RPLPO), while others show no issue. I carried out the RNA extraction and cDNA synthesis for the first set of samples which show a single peak, whereas the other two sets of samples processed by another in our lab give a double peak melt curve. The CT values for these two sets are around 1.8 lower than my set. The samples are all at the same dilution and the noRT controls have CTs >35. I've run this twice now and get the same result. Attached are images of the melt curve, the CT values and the products on 2% agarose gel. Are there any suggestions why these differences might occur?
Dear Sarah, I think there are unspecific amplifications or contamination in set2 and 3. Contamination of cDNA or primers. Your set 1 is clear in agarose gel and there is one peak in Tm analysis. So that, there is a problem in set 2 and 3. Set 1 Cp values are good and around 20.
So, if I understood well, if you do qPCR of the same three sets of cDNA samples but for a different target transcript, this problem does not happen, right? IOW, the problem is specific for bovine RPLPO, is that correct?
@ Mert Sudagidan, if this was a contamination problem it would have been picked up by her noRT controls.
Hi! Do you expect that set 2 and 3 have less expression of the gene? I think it is likely that expression of the gene is low in set 2 and 3. There might not be enough template so the primer ends up binding in other regions with affinity so this is why you observe on those sample the unspecific products and that the band from the target gene is weaker. So, to avoid this unspecific products, then it will be necessary to improve primer design. This is what i think that might be happening, hope this helps. You can try also to increase the annealing temperature to see if this helps to decrease the amplification of this unspecific bands.
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