After some “normal” experiments, unexpectedly in one group of samples during PCR amplifications Ct values are very low (in the range of 7-9) and thus we are getting very high amount of calculated mRNA in them. At the same time the housekeeper is in the normal range (Ct around 15-18). I have a doubt that at initial stages in these samples there is a “pseudo peak’ which machine accepts as Ct (see the amplification plot) and in reality amplification starts much later. I am also attaching amplification plot of housekeep.
Would be most grateful for your advices
There is a late amplification only for one primer (GADPH). As for target gene (ENS 89), it is not amplifying in no template control.
Thank you Lela Chitadze I was just checking that this was not a simple contamination issue
I would generate a serial dilution standard curve for that primer and see what the efficiency looks like. perhaps prepare a new tube of primer as well and test the current tube and the new tube. this also looks like a baseline issue in the software - but i'm not the one to answer that - technical service for the machine could help
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