Analyzing fold change when Cq value in treatment condition is 0 (not expressed)

Question

[jajarosli]

Hi everyone,

I conducted RT-qPCR to validate my transcriptome analysis. In my transcriptome data, the genes are expressed in Stage 1, but are not expressed in Stage 2. When I conducted qPCR, I was able to see the same pattern. However, I am facing problem when trying to analyze my data because my Cq value for Stage 2 is zero.How do I calculate my fold change for qPCR when my Cq values for Stage 2 is zero. I've used the readily available excel template and got huge positive number. Appreciate all the help I can get! Thank you!

[jared.andrews07]

Well, if it's zero, you can't show a fold change. Better off just showing the Cq values, gets the same idea cross. Just be sure to show them for your reference gene as well.

Edit: Devon's answer is an even better idea.

[jajarosli]

Thank you so much for the suggestion!

Answer 1

[Devon Ryan]

The Cq for an unexpressed gene is the maximum number of cycles that the qPCR machine goes through (for the ones I've used it's been 40), so use that. This will underestimate the actual fold-change, but that's fine.

[jajarosli]

That's a great suggestion! Thank you so much. Do you know of any paper that applied this method?

[Devon Ryan]

In my experience qPCR machines return the number of cycles by default if there's insufficient signal, so I suspect a lot of papers have done this unknowingly.