hello every body
repeating RT-PCR run using the same cDNA way would the CT value for hUBC ( endogenous control) change ?!! this change the CT value affect the result?and give opposite result each time !!
I am using RT comparative delta CT to find the relative expression on gene !
please help :"(
Plate to plate variation(due to various reasons) is normal in realtime PCR, so it is always recommended to to run endogenous control and gene of interest for same sample in single plate so that the variation of these two gene should be same.
Good luck..
It might be because of the different amount of cDNAs you are adding each time. By different amount, I mean differences in nanoliter and not microliter. It normally happens and therefore don't worry!
Because of the high sensitivity of qPCR technique, this negligible difference between the materials can result in a huge difference. But since you are evaluating endogenous gene in all your experiments the changed Ct value is normalized to that and your calculations become true. I mean, if Ct value of UBC changes from 21 to 19, the Ct value of your target gene is also automatically decreased accordingly, and delta Ct is 2 for both experiments.
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