Hi,

I have done 2 qPCRs for 3 different targets and 1 reference genes. All my samples come from different patients. These are not paired samples. I have a calibrator category of sample with which I would like to compare other samples from different tissues. My set up is as follows:

1. 3 different patient samples from 3 different tissues (A,B,C; n=9) in each experiment, 3 samples from tissue A is taken as the calibrator.

2. I have done individual statistics. Now, I'm trying combine the results to analyse all the samples. So, I have 6 samples from 3 tissues (A,B,C).

3. I have run standard curves both the times to check for target amplification efficiencies. They look agreeable.

My question: I'm trying to average all the 6 calibrator samples to consider as a single value when calculating the fold differences of my targets for the other tissues(B & C). This gives quite a big range of fold differences between the samples from the same tissue. Am I doing this right? If not, please tell me how to go about this. Also, what kind of a statistical test should I apply to test the significance of fold differences of my targets across tissues?

Many thanks!