Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
- - - - -

qRT-PCR calculation question

  • Please log in to reply
No replies to this topic

#1 schmidthead



  • Members
  • Pip
  • 1 posts

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?


Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.