I want to ask about Pfaffl model for relative quantification. I use the formula:
ratio = E target^(dCt target)/ E reference ^(dCt reference).
Lets say I use normal cells for the calibrator, so the dCt is Ct differences between the normal cells and the samples, right? So what is the result? Is it n-fold relative to reference gene or is it n-fold relative to normal cells?
And I got question about the calibrator. If I run an experiment to comparing pre- and post-treatment expression for 10 patients (each patient has both pre- and post-treatment tissue, so in total 20 samples), is it better if I use the pre-treatment tissue (tumor tissue) or normal tissue as the calibrator?
Really need your help through this. Thank you