I am working on HTqPCR miRNA data gathered from the OpenArray platform. By default Ct values for those probes which do not cross the threshold fluorescence is set to 40, as that is the number of cycles which the machine goes through. However I notice that out of ~750 miRs by 72 samples there are only a handful of outliers reaching slightly over 35. As the samples have undergone an amplification step I do not think the Ct value can be related to an actual number of copies in the sample. My thinking is that over 72*750 miRs if no result is over a set value then it is likely the value represents an 'effective zero' or a lose approximation. Therefore I was thinking of taking my maximum Ct as being 35.
Is my approach reasonable?
A LifeTech (who now own OpenArray) employee confirmed that generally taking a Ct value lower than 40 is a reasonable thing to do and said that there was ongoing academic debate about it, though has not been able to provide me with any links/literature/suggestions.
I have come across a couple of posts on forums discussing what an appropriate cut-off might be. 'Wozzles' talks about 35 but doesn't mention where he got it from and the only reply with an explanation isn't a helpful approach to a high-throughput problem. Other posts are similar (in fact I came across Wozzles asking the same thing again elsewhere!). I have also just now found a paper mentioning the problem, but focussing mainly on outlier-calling.
