I am trying to use QPCR to determine DNA copy number of a transgene as it is suspected to have dropped within a colony. So far I've used the comparative method of quantification rather than absolute method. I'm carrying out duplex reactions on an ABI 7500 system. My control sample has been extracted using a different method to my test samples.
I am having difficulty working out how to analyse my data as the level at which I set the threshold is having a massive impact on the results. The baseline setting also seems to have a large impact on the results, even when it is set within the apparent baseline and even when only small changes are made to the settings.
I understand that I should try and set the threshold to the lowest possible point during the exponential phase. This varies a lot between samples. I've tried setting it according to this rule but even slight alterations seem to have big implications in the results. I think this might have something to do with the DNAs being extracted using different methods and the effect it has on the PCR efficiency. I've looked up methods of calculating efficiency and standardising for this, but these all seem to refer back to the Ct, which of course depends on the threshold I have set.
Any help on this would be really appreciated. I feel like I'm going round in circles!