Hi, I'm working on qRT-PCR data analysis to validate the result of the microarray analysis. The qRT-PCR ran 45 cycles to get the CT value and each sample was performed in triplicate. However, the CT values were Undetermined, 26.96, and 26.92 in one sample. Some studies set the CT value of the Undetermined as 40 mostly. So I should set the CT value as 45 (because we ran 45 cycles) and then calculate the CT value of this sample by (45+26.96+26.92)/3 = 32.96. Is it right? or should I just ignore the Undetermined and calculate the CT value by (26.96+26.92)/2 = 26.94?
Another question is I found there is no consensus on defining whether a gene was validated by qRT-PCR. Some studies mentioned if the direction (up-regulation/ down-regulation) is the same in microarray and qRT-PCR and p<0.05 in testing, then this gene is validated. Is there any guide to follow?
Thank you.
