I am new to the forums and hope I can find some answers to my issues, nobody in this lab seems to be doing qPCR.
So I am interested in looking at changes in expression of one gene, I have about 400 samples, I ran three references genes and all I want is a d(ct) value for each sample representing the level of expression of the gene.
So I don't have any calibrator just the reference and the target gene.
As my references and target have different efficiency, I decided to use the Pfaffl method. So my d(ct) = Efficiency target ^ ct target / Average efficiency ref ^ ct ref
However, here is my issue, using this formula I end up with high dct value when the ct value are actually high. In brief the dct values are kinda of reverse. The samples were expression is higher give me a lower dct.
So when I try to plot everything for publication it is all reversed and then the graph does nor make much sense anymore.
Does anyone have any suggestions for my problem, maybe there is smtg in the calculations I am doing wrong
Thanks a lot for your help