Endpoint RT-PCR for quantification of mRNA?
paul_wary at yahoo.com
paul_wary at yahoo.com
Thu Jun 1 04:03:34 EST 2006
Austin P. So (Hae-Jin) wrote:
> 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 was thinking about this again and you are absolutely right. But
assuming that the efficiencies for the reverse transcription of the
housekeeping gene and the other genes of interest are approximately
equal, the housekeeping genes should also provide some normalization
for the reverse transcription step (after all, it is also used for this
purpose in real-time RT-PCR, isn't it?). Yet, whether this assumption
is true is difficult to determine.
> 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.
OK, I interpret this in the way that there is no such thing as an
"end-point" PCR for quantification of mRNA if "end-point" means
amplification until the PCR hits the plateau phase. You have to stay
within the exponential amplification phase. There is no correlation
between the height of the plateau ("end-point") and the intial amount
of cDNA, right?
How low should the cycle number be? I know this depends on the initial
amount of cDNA; with less cDNA the cycle number can be higher without
reaching the plateau, but are there any estimates?
> 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).
Does the type of DNA detection method matter for the quantification? In
this case, the gel was stained with ethidium bromide (EtBr) and the
bands were quantitated densitometrically. Does the intensity of EtBr
stained bands correlate well with the amount of DNA in a gel band?
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