Quantitative PCR

Frank Chen yatsen at wam.umd.edu
Thu Aug 31 14:28:17 EST 1995

	The one that argues for it always says: This has appeared in such 
such publications and now patents, or this has been highly evaluated by 
reviewers.  What is the scientific evidence? - Controls?
	The one argues against also has some sort of scientific 
arguments.  For example, Dr. Miller's points in his reply are worth 
citing here.
	I will give you my simple controls.  I frequently did two 
"identical" PCR reactions starting with the "same" amount of templates.  
Most likely, I got different amplifications.  Which one had more template to 
start with?
	Suppose you have two samples for quantitative analysis by RT-PCR, 
one gives you stronger signal than the other?  Are you going to be 95% 
sure that there is more specific mRNA in the one that has stronger signal?
I will hesitate to conclude myself.  However, I will be quite confident 
to conclude such a result if I repeat 3 times by convential Northern 
and/or insitu analysis.

	Sorry, if this offends you.

Frank Chen

On Thu, 31 Aug 1995, William Alexander wrote:

> In Article <41vo24$ok2 at miasun.med.miami.edu>, tmiller at newssun.med.miami.edu
> (Todd Miller - Pharmacology) wrote:
> >In article <thomas.kreuzer.62.0012F0A2 at kfunigraz.ac.at>,
> >Thomas Kreuzer <thomas.kreuzer at kfunigraz.ac.at> wrote:
> >>On Thu. 24 Aug Frank Chen wrote as a reply to a question concerning 
> >>quantitative RT-PCR:
> >>
> >>" No matter how you do it and how good your controls are , your results from 
> >>quantitative PCR will be questionable."
> >>
> >>Since I am trying to put up a quantitative RT-PCR - method myself (and being 
> >>quite new to the method of PCR in common) I would be interested in the major 
> >>reservations against the principle of quantitative PCR and especially 
> >>quantitative RT-PCR. What exactly makes itYs results questionable and why 
> >>canYt these problems be avoided ?
> >>It would be a great help if you could also give me some advice about 
> >>literature dealing with these problems.
> >>
> >I would highly recommend reading an article by Luc Raeymaekers in
> >_Analytical Biochemistry_ (I think it is volume 214, 1993, but you
> >may have to check your favorite citation source).  He goes through
> >all the mathematical theory of quantitative competitive PCR and comes
> >to the conclusion that there are many papers published that have
> >ignored some basic theory and predictions, which makes the data
> >very suspicious.  Basically, the assumption that your competitor and
> >your target have equal amplification efficiencies is almost never
> >borne out, and this results in huge errors by the time you go through
> >all the cycles.  
> >
> >Quantitative PCR works only for HIV and AIDS patients...  ;-)
> >
> >Todd Miller, PhD
> >University of Miami School of Medicine
> >tmiller at newssun.med.miami.edu
> >
> > 
> >>Thanks in advance,
> >>
> >>Tom Kreuzer
> >
> >
> Todd Miller, Did you read Isaac Kim's Post?  He is correct (great arguments
> and the experiments were done) and so is Tracy.  Quantitative PCR does work,
> you should not use a "house-keeping" gene (GAPDH) as your competitor and the
> source of the nucleic acids has nothing to do with this topic.  
> Todd said:
> Basically, the assumption that your competitor and
> >your target have equal amplification efficiencies is almost never
> >borne out, and this results in huge errors by the time you go through
> >all the cycles.  
> The whole idea is to design the competitor so that it does amplify at the
> same efficiency as the target.   (See Celi et al., "A rapid and versatile
> method to synthesize internal standards for competitive PCR" Nucleic Acids
> Research, 1993, Vol. 21, No.4, 1047.)  Yes, Todd is also correct, if the two
> do not amplify at the same rate, the paper should be ignored.  
> There are at least two simple ways to design good competitors:
> 1.  Use the "same" product as your competitor by adding or removing a
> restriction site so that the two products can be distinguished an a gel
> after restriction.  (This extra restriction step can be a pain.)
> 2.  Use a competitor that is slightly different in size to the original
> target so that the only sequence difference is this extra Nucleic acid
> fragment. 
> This is the case where Todd gets caught up in mathematics.  The enzymes used
> for the amplification are extremely fast (do you need references?).  
> Therefore, the size difference can be designed so that it is detectable on a
> gel but does not change the efficiency of the amplification of the target
> and the competitor.
> Two US Patents (there may be more) have been issued in this area.
> 1. Wang et al., Patent # 5,219,727, Date of Patent:  June 15, 1993, Filed:
> Sep. 28, 1989; "Quantitation of Nucleic acids using the polymerase chain
> reaction"
> 2. Bunn et al., Patent # 5,213,961, Date of Patent:  May 25, 1993, Filed:
> Aug. 31, 1989; "Accurate quantitation of RNA and DNA by competitive
> polymerase chain reaction"
> These patents are great scientific papers and should be available around the
> country (Frank Chen, U of MD College Park should have them in their lib.). 
> As you can see, they were both filed in 1989 and issued in 1993. They are
> both too "old" to be included in the full text biotech patents available on
> the WWW.  (See:
> http://www.inform.umd.edu:8080/EdRes/Topic/AgrEnv/Biotech/Biotechnology_Pate
> nts-full_text)
> I just don't think this area is so new anymore.
> Bill Alexander
> alexanderw at cber.cber.fda.gov

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