I need to measure the amount of endogenous mRNA from a transgenic strain that produces endogenous and exogenous mRNA. I am able to distinguish exogenous mRNA by using a forward primer specific to GFP which is fused to the 5' end of the transcript. Thus, I can run two reactions, one that will only detect the exogenous transcript, and one reaction that will detect both.
I would like to use qRT-PCR to determine the % of mRNA that is exogenous and the % that is endogenous, but am unsure how to calculate this. I imagine I could use an absolute quantification to do this, but is there anyway I could calculate this with a variation on the relative quantification? I have primer efficiencies for both sets of reactions, and since I am measuring the same sample, can I just calculate this with say,
efficiency exogenous^(Ct exogenous)/efficiency endogenous^(Ct endogenous)
Would this be a valid way to do this, or does anyone know how this can be done?
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qRT-PCR calculation question
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