Dear All,

There is a question always bothered me when I am calculation gene expression level by using Ct value, so lets say, you have three value of Ct for housekeeping gene, and you have three values of Ct for the target gene, lets say its like this:

Ct house1=24.5; Ct house2=25.3; Ct house3=40.2;

Ct target1=30.1; Ct target2=33.0; Ct target3=35.6;

so, for housekeeping gene and target gene, I have triplicate for both. How do you calculate your delta value? do you make an average for all the houseCt value first, and then you make an average for your target gene, then you do 2^(house-target) or,

do you do house1-traget1, then you calculate it three times, and then you get the value?

The other question I have is that, say I run the triplicate different day, and for day 1, the software tell me that the cutoff value is 11, then it give me a Ct value based on the automatic baseline, however, I repeated the qPCR again day 2, and the software tell me that the cutoff value is 13, then it give me the other set of Ct value. Should I manually change the baseline the same for all different days or should I do what the software said and do automatically ?

how do you exactly calculate the Ct value if you have more than one sample? and triplicate or more? how do you pick the baseline?