I'm conducting a time course study on gene expression using RT-q PCR for samples treated with 4 conditions: vehicle, RA agonist, Calcitriol agonist or a combination of both agonists over 6 hours. I'm expecting to observe a gradual increase in expression over time for the combined treatment condition due to an additive effect of the ligands. Indeed, I have observed that for all of the time points except for the last one where the Ct value for my combined treatment is 30 while my untreated control at zero hour has a Ct value of around 28.85. Even the Ct value for the vehicle condition for my last time point is around 28.65 so, why am I getting such Ct for the combined treatment?
The Ct value depends not only on the gene expression but also on the amount of biological material used, its quality (integrity), assay parameters (e.g. background correction, selection of a threshold value) and the assay performance, all of which can differ between samples and/or runs. Only after somehow controlling all these possible influences you may interpret differences in observed Ct values as differences in gene expression. This is typically achieved (at least in part) by measuring the CT values of some internal control with presumably constant expression under all conditions and in all samples and using plate calibrators where measurements should be compared across plates or runs. And further, Ct values can be quite variable between samples, what mean that it needs some statistics check the statistical significance of observed differences.
Given all this was done but just not communicate by you, then the observed result may indicate a counter-regulation. If so, it should also dampen at even later time-points.
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