A quick question - I am running a basic qPCR experiment and analysing by the delta ct method (comparing target gene to endo control). However, I am wondering about the threshold level settings. By default, the software sets the threshold value at 0.2 in both cases. However, this is not an ideal threshold. When I click 'auto' threshold, they do change, but the values are different for my target gene versus my control gene. Does this discrepancy in thresholds matter? I know it would affect the final delta Ct value so am I better manually setting or letting the auto function do it's thing?
Thanks a million