Endpoint RT-PCR for quantification of mRNA?
Austin P. So (Hae-Jin)
nobody at nowhere.com
Thu Jun 1 17:24:12 EST 2006
paul_wary at yahoo.com wrote:
> 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?
I was given some invaluable advice on the qPCR list-serv, and basically,
a way to estimate it is that you don't want to reach a point where the
reagents (primer, dNTPs) become limiting, and the rule of thumb that was
given was to ensure that your final product was 1000-fold less than the
lowest concentration of reagent.
So for example, if your PCR mix has 1 microM primer, and 0.2 mM dNTP,
you don't want to have your amplification product exceed 1 nM (based on
primer concentration). If your starting concentration is 0.1 pM (~6 ng
of a 100 bp template), then a 10,000-fold amplification is ~13-14
cycles. If your starting concentration is 0.1 fM (~6 pg of a 100 bp
template), then a 10,000,000-fold amplification is ~23-24 cycles.
This is only if you are working with quantified DNA, and as Duncan
noted, there are a host of other issues that confound this, particularly
when you are starting from RNA. But you can make some general
assumptions of cDNA yield (it ain't an exact science anyway)...
The limitations to using a gel-based assay is not so much due to the
sensitivity of the dye, but more so the densitometric method used as
well as the fact that you are using a fixed number of cycles across all
your samples. If you take a really really nice picture and scan the
film, you can get a 16-bit TIFF image, which gives you a dynamic range
of 0-65536. If you use a CCD, you are restricted by the recorded image
(8 bit = 0-256, or 16-bit = 0-65536). So if you are lucky, you may be
able to see 3-4 orders of magnitude. But then you run into the problem
of using fixed cycles...
This is different than using a real-time assay, since you are not
limited to applying a fixed number of cycles to each sample, so you can
get to 6-7 orders with due diligence.
Hope that helps...
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