Endpoint RT-PCR for quantification of mRNA?

paul_wary at yahoo.com paul_wary at yahoo.com
Wed May 31 15:57:58 EST 2006


Hi,

In a Ph.D. thesis I read, the author used endpoint RT-PCR in order to
quantitate relative amounts of mRNA. I asked myself whether this is
actually possible. What the person did was this:
1) Take equal amounts of mRNA from different sources that were supposed
to be compared.
2) Reverse-transcribe the mRNA in parallel.
3) Amplify equal amounts of the resulting cDNA in parallel.
4) Separate the PCR products with a gel, stain the bands and compare
their intensity in order to obtain the ratio of mRNA levels in the
different sources.

As far as I know even under apparently equal or very similar conditions
the variations in efficiency of reverse transcription and amplification
are too big to allow quanititating mRNA levels that way. And using a
housekeeping gene for normalization of the PCR step would still be
insufficient because there is also variation in the reverse
transcription step.

Isn't this why real-time RT-PCR is actually the adequate method for
this purpose nowadays? I know of competitive RT-PCR assays but this is
different. There is no competition anywhere as far as I can see.

So, my question basically is: Is there such a thing as a
semi-quantitative endpoint RT-PCR?

Paul



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