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