I'm quite new to doing qPCR and after optimising my target of interest and housekeeper primers, I've ended up with them having differing optimal annealing temperatures. How do I run my actual samples with these when they are 2 different temperatures?
Well, it depends on how fancy your QPCR machine is. Mine has a single block with a single anneal/extend setting. Some machines can run a gradient, which means you could set up your housekeeping and target samples at their ideal temperature.
For me, I'd have to optimizer the primers so they work at the same temperature (this would likely involve lengthening the primers with the lower Tm to bring it up to match the other set)
What sort of setup is possible for you?
Run your gene expression assays in separate plates. House keeping gene(s) in one set of plates at the required temperature to get 100% efficiency. Target genes in separate set of plates at each of their respected optimal temps to maintain 100% Efficency. You will sacrifice compenstaing for plate to plate variation since doing it this way obviates including calibrator samples. However if there is a standard curve included in each plate it should give good results in the end.
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