Hello, I ran an experiment in order to check if a gene was been expressed in a circadian pattern. In addition I ran the some classic circadian genes (Clock, Bmal1, Cry1, Cry2, Per1, Per2).
Now I have a question about the analysis, when you analyse qPCR you need to have a "control" group or baseline time point to compare against. In this case I don´t, right? Because which time point should I selected to be my baseline or control in circadian gene expression?
Then, if I don´t have a control, how do I calculate 2−ΔΔCt value?
Thank you very much for the help.
Best regards
Marcela
You don't have a "control".
There is no need to calculate ΔΔCt values. To show/explore/analyze the circarian rhythm it is enough to have ΔCt values. You can plot the ΔCt values against the time of the day and fit a sinusoidal model. The amplitude will be sensible estimate of the ΔΔCt value, indicating how much the expression changes maximally during a day. Another option is to give the root mean square of the amplitude (see picture).
Edit: I forgot to explain the formula, but as you analyze wavy function you should know that. Anyway: T obviousely is the time span of a wavelength (that is: a day), and f is the wave function, giving the ΔCt values depending on the time.
Add-on: I just realized that it might be a good idea to use the mean ΔCt as the "control", that is, to shift the curve vertically so that it oscillated around zero. The curve is then a curve of ΔΔCt values, each value shows the difference to the average expression.
You don't have a "control".
There is no need to calculate ΔΔCt values. To show/explore/analyze the circarian rhythm it is enough to have ΔCt values. You can plot the ΔCt values against the time of the day and fit a sinusoidal model. The amplitude will be sensible estimate of the ΔΔCt value, indicating how much the expression changes maximally during a day. Another option is to give the root mean square of the amplitude (see picture).
Edit: I forgot to explain the formula, but as you analyze wavy function you should know that. Anyway: T obviousely is the time span of a wavelength (that is: a day), and f is the wave function, giving the ΔCt values depending on the time.
Add-on: I just realized that it might be a good idea to use the mean ΔCt as the "control", that is, to shift the curve vertically so that it oscillated around zero. The curve is then a curve of ΔΔCt values, each value shows the difference to the average expression.
Hello Jochen Wilhelm ,
please can you give reference for that it might be useful for us.
Thanks
Rajesh
Dear Rajesh,
I don't know for want kind of references you are looking for. I don't have any specific references for analysing circardian gene expression. There are plenty of books about the analysis of gene expression as well as of the analysis of periodic functions, all of which will be easy to find in google.
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