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qRT-PCR calculation question


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#1 schmidthead

schmidthead

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Posted 22 April 2010 - 12:02 PM

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?

Thanks.




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