Semi-quantitative PCR

Martin Offterdinger martin.offterdinger at akh-wien.ac.at
Wed Jul 26 04:34:11 EST 2000


Tina <anfortin at webnet.qc.ca> schrieb in im Newsbeitrag:
Tk4f5.1080$WM6.81746836 at news1.mtl.metronet.ca...
>
Dear Tina,
I once used competitive RT-PCR. You need to have an in vitro transcribed
cRNA that  can be amplified using the same primers as your gene of interest.
The amplification product of this cRNA has to have eiher a different size or
at least a different restriction site. This maens that you have to produce
such a cRNA first. Afterwards everything is rather simple. You keep the
amount of total RNA (from cells!) constant an´d add different amounts of
cRNA. So the cRNA and you cellular RNA compete for primer binding. If the
relative band intensities of the cRNA PCR product and your cellular PCR
product are equal you have the same amount of RNA present in your cells as
in the added cRNA.
You dont need to worry about PCR plateau etc.. as this relationship holds
true for all stages of PCR. If you assume that the cRNA and your cellular
RNA are amplified with the same efficiency, you can even get an absolute
quantitation of your RNA.
Hope this is of some interest to you.
Martin







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