I am using a biorad CFX machine to do real-time PCR. I am trying to validate candidates for a reference gene.
In their program, the threshold can be calculated for each target (primer set) or by fluorophore (everything).
It does make a difference in the analysis.
I am weighing the pros and cons of each. I am leaning towards target-specific thresholds because their profiles can vary by a few percent in efficiency, sigmoidal adherence, etc. But I can't find information on which to use and why there is a difference in the first place.
What do you suggest?
Hey... checking whether this works or not... it does (sometimes this site does not work for me).. Go for target-specific threshold always.
Thanks, Jack, for the response and the excel sheet. I saw mention of efficiency adjusted calculations for relative comparison but your document really puts it in perspective. I appreciate the equations!
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