I am analyzing temporal changes in target gene(s) expression during embryonic and juvenile (fish) development using qPCR.
Since I don't have a control group, the usual fold change calculation methods (Pfaffl, deltadelta...) don't apply.
I guess could calculate absolute expression levels, but since I'm mostly interested in temporal changes in expression, I was wondering if there is an acceptable way to calculate relative expression in relation to a housekeeping gene (beta-actin).
Hello, as long as you can prove that your reference gene(s) are stable across development, relative expression sounds like a fair approach to me. What you could do is, after normalising against the reference, normalise again against time 0.
That said, bear in mind that if you normalise against time0 you cannot do any comparisons between the expression of different targets. If you wanted to compare between targets, you should do absolute expression.
Cheers,
Ruben
Thanks, Ruben!
There are some publications dealing with the expression of my reference gene in a related fish species. Guess I'd have to call it a "proof".
Hello,
In the past few days assumptions have fooled me (e.g. I assumed that a plasmid had a single restriction site for an enzyme and troubleshooted accordingly, but it actually had two restriction sites).
In your case, assuming that that gene in a related species is a reference might give you the wrong results -if it is actually not a reference.
What I would do is take that candidate to reference gene and a few others that you think could be candidates, and use a geNorm approach (https://genorm.cmgg.be/) to determine their stability across your conditions.
Paper-wise, you could then think of publishing a small note on the selection of reference genes and then the main paper on your gene of interest, or a full paper with everything.
Cheers,
Ruben
The gene has been used extensively in my species as a reference, but not for embryo development.
Looking into geNorm now.
Thanks for the recommendation.
Cheers,
Ivan
Yep, be wary then, just in case. Sometimes the reference is stable in some conditions, but in others can vary a lot! It's just that it happens, and I rather invest time at the beginning than throw away data at the end :)
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