Please excuse my basic questions, but I'm quite new to qPCR and trying to learn as I go.

One of the experiments I am carrying out is to compare the expression of Gene X between two samples (disease positive vs. disease negative). I have carried out qPCR for the gene of interest, as well as an endogenous control (GAPDH in this case) and generated Ct values at an appropriate threshold. The average Ct for GAPDH in samples 1 and 2 was within 0.2 of each other.

From here, please correct me where I deviate from correct protocol, or show misunderstanding ........

- I first calculated the average, standard deviation and %RSD for the replicates of each reaction.
- Next, calculated the deltaCt of Gene X for each of samples 1 and 2, by subtracting the Ct for GAPDH from that of Gene X. This gave me a deltaCt for each of sample 1 and 2.
- Calculated Relative Expression for samples 1 and 2 using the equation (2^-dCt). This gives me a value representing the expression level of Gene X in each sample. These values may then be compared to each other as relative expression values between samples 1 and 2.

I get a bit muddled from here on .....

- I was then asked to calculate the delta.deltaCt by subtracting the deltaCt of sample 1 (disease) from that of sample 2 (non-disease).
- Calculate a comparative expression using the equation (2^-ddCt). This gives me a single number indicating the expression of Gene X between samples 1 and 2.

Is the logic of all this correct or have I misunderstood or blatantly got everything wrong?

Many thanks to all for input.