Calibrator sample has zero expression of target gene.
Posted 03 October 2013 - 05:10 PM
I am currently working on optimising a qPCR for relative gene expression of a cytokine using the 2-^dd Ct method. I have so far used untreated sample at time point 0 hr as the calibrator. However, for this particular cytokine there is no gene expression in untreated sample at time point zero. So when applying the above mentioned formula what, ct value should I use. Zero or the last cycle of the run which is 35?
Posted 04 October 2013 - 06:56 AM
Are you trying to compare the fold increase of your cytokine at time point zero vs. a later time point?
There are a couple of different ways you can approach your quantification depending on what you are trying to show. Does the treatment of your sample suddenly switch on your cytokine or is your cytokine usually differentially expressed? Are you trying to compare your cytokine expression to the expression of a common ubiquitous cytokine?