Endpoint RT-PCR for quantification of mRNA?

Austin P. So (Hae-Jin) nobody at nowhere.com
Wed May 31 16:20:35 EST 2006


paul_wary at yahoo.com wrote:

> 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.

These issues you bring up apply whether or not you do normal PCR or 
real-time PCR.

I think the thing to keep in mind is that there is no fundamental 
difference between real-time PCR or just regular "end-point" PCR, 
particularly if you do not hit the plateau phase of the reaction by 
keeping the cycle number low, and you are interrogating a single transcript.

The advantage of real-time is that you do not have to run a gel, and it 
doesn't matter if you extend your run beyond the plateau phase, because 
you have all the low-cycle information stored already to get relative 
abundances. You can also get "absolute" levels by having a standard 
curve (which you could also do using an end-point method, but it would 
be tedious as hell).

Austin


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



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