I am new in qPCR, sorry if the question is very basic. I will run B-actin as a reference gene, but the anneal temperature for this gene is different than from Leptin, the gene I am interested in, and in the conventional PCR I run B-actin for 30 cycles and Leptin for 35. Do I have to run them separately? If yes, how I compare them?
Dear Juliana,
have a look at the attached files. These will give you a general introduction to RT-qPCR. This will explain a few things, such as how to validate your reference genes (ideally you would use more than one) and should also help you to understand the answer to your question.
There is no problem in running the two PCRs separately (they are two separate PCRs anyway!) and you are doing relative quantification. As long as you amplification efficiencies for both PCRs are close to 100%. You are using the reference genes to compensate for differences in RT-amplification, pipetting, RNA quantity and so on between your samples. Ideally the reference gene expression levels don't change between your treatment and control - however, you can't just assume that this is the case, therefore you need to validate your reference genes first.
Also, actin may not be your best option as it is fairly highly expressed in the cell and leptin probably quite a bit less. Ideally you would have reference genes that are fairly similarly expressed...
I think Petra's answer is a good one and generally I agree with what has been said
My only slight caveat would be that if you perform real time PCR you ideally need your housekeeper and test sample on the same plate and thus subject to a common set of PCR conditions. This is because you can experience intra assay/ plate variation although I do agree with Petra that significant differences are more about sample quality and/or quantity
Thus, next time I would suggest redesigning primers and alighting on a common Tm for both housekeeper and test gene but in the mean time do as Petra has suggested
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