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Untreated samples negative, how to analyze fold change?

fold change untreated analysis

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#1 AnneUW



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Posted 29 January 2014 - 02:31 PM

I'm trying to analyze a set of data.  For one gene, the untreated samples are basically negative and are very close to the NTCs' Ct values.  The treated samples have a very good increase in expression.  Following my usual policy of not using data within 5 Ct of the negative control, I used a new primer set on the same cDNA but got a similar result.  Is there a way to use this data and phrase the analysis in a way to make it clear that the fold change is not exact since the untreated samples have such low expression?  I'm sure this happens, but I am having trouble phrasing a search to bring up published papers that have had this issue.

#2 Trof


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Posted 03 February 2014 - 03:11 AM

I've seen the way of analyzing negative data as using the "number of cycles in the program"+1 as a 'Ct' for the negative calibrator. However I don't have any reference for that (I heard it on a qPCR conference and didn't make notes) and I even think I was looking for it on several occasions.

You could mention it under the results table, how you acquired calibrator Ct for this analysis.


But generaly is better to use a positive sample as a calibrator, but some genes can have virtually zero expression in normal or control/untreated samples, eventhough other genes are normalized well. The aforementiond method is used exactly in this case, when you can't get a better calibrator or/and it's a part of a larger analysis set.

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