I hope this has not been covered already - but I'll ask anyway.
I am looking at expression of a gene in bacteria using 2 step RT-qPCR. Recently I had issues with the standard curve for my target gene showing multiple peaks in the melting curve toward the lower template concentrations. I raised the annealing temperature which seemed to fix it.
However, now I am trying to develop primers for an endogenous control, and it looks like the annealing temp of primers for my target gene will be too high for my endogenous control primers.
Do you think I can run one PCR plate for my target gene and then another for my endogenous control at different annealing temps? - or will that bring in too many variables?
I have also thought about trying touchdown PCR to incorporate both temps ..?
Any suggestions would be greatly appreciated!
Primer sets with different annealing temps??
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