real time PCR

Rui Pires Martins rmartins at genetics.wayne.edu
Thu Jul 5 13:30:12 EST 2001


a question for anyone who works with any sort of rt (real time,
flurorescent) PCR...

we're trying to use rtPCR quantitatively on fractionated DNA  to see
whether a fragment is more likely to amplify in one franction vs.
another.  the curves that are generated (sigmoid, asymptotic) are
generally coming up at pretty much the same cycle, ie. both fractions
will begin to show increasing fluorescence at say, cycle 25, but one
will inevitably plateau sooner (ie. not high) as another...therefore one
reaction will yield more net product than the other.  Now this isn't
always the case, sometimes one reaction will not only come up higher,
but also begin the linear phase of the curve before the other sample.
My question is then, when (quantitatively) declaring a fragment more
prevalent in one fraction over another, is the critical parameter the
maximum fluorescence of the sample, or should the cycle at which the
sample starts to amplify also be considered?  Secondly, it has been
suggested to me that to account for both of these parameters, I should
use the cycle number at which the middle fluorescence value for that
curve is achieved (an cycle#(f50), if you will)...any thoughts?

thanks for your time and consideration.

cheers,

rui

--
Rui Pires Martins

Wayne State University School of Medicine
Center for Molecular Medicine and Genetics

rmartins at genetics.wayne.edu






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