Methods and reagents: Quantitative PCR: an accurate measure of mRNA?
by Paul N. Hengen, Ph.D. *
Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet. This month's column discusses quantitative measurements of messenger
RNA levels by using reverse transcription polymerase chain reaction (RT-PCR).
For details on how to partake in the newsgroup, see the accompanying box.
Quantitation of gene expression requires that an accurate measurement of a
specific transcript is made, and netters recently had a battle over whether the
polymerase chain reaction (PCR) can be used for this.
One technique, called reverse transcription polymerase chain reaction (RT-PCR),
has become much more popular of late. RT-PCR is a multiple-step PCR
accomplished by isolating total RNA from a cell line, using reverse
transcriptase to create a pool of cDNA, and then amplifying a predetermined DNA
fragment through the use of specific primers and thermocycling. The amount of
a specific gene transcript can theoretically be measured by estimating the
intensity of the amplified DNA band on an ethidium-bromide-stained agarose gel
or X-ray film by densitometry.
In comparison to a standard co-amplified mRNA control sequence, the band
intensity is used to estimate the initial amount of mRNA available for the
reaction and, thus, the amount of gene expression from the particular gene
of interest.
Some netters were recently complaining that although the use of PCR for
quantitation has been uncritically accepted by many scientists, it really
cannot be relied upon for quantitative measurements of nucleic acids owing to
the very nature of the amplification process.
When DNA is amplified by PCR, the efficiency of each extension reaction per
cycle is not always 100%. Given an initial amount of template and more than 20
cycles to complete the PCR, the amount of DNA product at the end of the
reaction is not always as much as one might expect. This is due to a plateau
effect after the exponential increase of product, which is dependent on the
initial amount of target. [1] One netter summarized what can be considered the
sentiments of many scientists when he said `No matter how you do it and how
good your controls are, your results from quantitative PCR will be
questionable.'
This prompted a discussion about what exactly makes the results of RT-PCR
questionable and how some problems inherent to the process might be avoided.
Back to basics
One person pointed out that an article by Luc Raeymaekers
(Luc.Raeymaekers@med.kuleuven.ac.be)
suggests that many have ignored some basic
theory and predictions. Raeymaekers [2] defines the amplification of DNA
within a RT-PCR in terms of a mathematical model, which can be used to explain
why quantitative PCR can lead many researchers to think that they have a handle
on gene expression levels in their experiments.
In a situation where the target sequence (T) and the standard sequence (S, the
competitor) would amplify with different efficiencies, the model reveals that
even the smallest difference between the efficiencies of amplification (E) can
lead to very different quantities of end products of T and S, which can result
in an erroneous estimate of the amount of initial material. The argument is
made that S and T sequences are often wrongfully assumed to have equal
amplification efficiencies, the result of which can be disastrous misjudgements
made from the amount of product seen on a gel after 20 to 30 amplification
cycles. [3]
According to netters, even with the most carefully designed primers, the two
efficiencies in most reactions involving co-amplified sequences are almost
never the same. An exception might be when the T and S sequences differ by a
single base pair, for example, as in the case of an engineered mRNA with a
restriction site planned within it so that the standard DNA amplified from it
can be distinguished from DNA amplified from the native mRNA, and both
templates are amplified in the same reaction using the same set of primers.
However, some people thought that Raeymaekers' model might be a poor one, not
being a completely true representation of the in vitro situation. They said
that if it cannot be confirmed through further molecular experimentation, then
the argument is moot.
Effective controls
How different are the efficiencies of amplification in different experiments,
and how might they be controlled or avoided? One solution would be to use a
more carefully selected control. Some people feel that a `housekeeping' gene,
i.e. one that is expressed in all cells and does not vary much in expression
during the cell cycle, should be used as competitor. But this is still
considered an external control that might suffer from the same problem. Another
idea is that, by using internal controls, the competitor could be designed so
that it amplifies at the same efficiency as the target. [4,5]
In addition, it was mentioned that a large number of cycles should not be run
to avoid taking any measurements after the plateau phase of the amplification
has been reached. Someone else proposed that, as an added precaution, serial
dilutions of the product should be made after the cycling to avoid the problem
of band saturation.
RT-PCR supporters
While some were calling for the need to create PCR police to help keep the
rampant misuse to a minimum, others felt that, as well as being accurate, it is
a well-accepted practice and the issues of this debate are old news. They think
that researchers should be able to use this technique without reservation
because the issues have already been thoroughly debated and there is no longer
any concern that RT-PCR is not a legitimate way to quantitate messenger.
Netters said that they have had little trouble publishing articles in which the
technique had been used, and that the reviewers agreed that the technique can
be used to successfully demonstrate levels of specific transcripts. They felt
that if rigorous conditions of the standard are carefully met, RT-PCR can be
more accurate than using a northern blot analysis.
RNase protection and northern blotting
Others disagreed completely, insisting that the older, but more time-consuming
methods of RNase protection assay (RPA) and northern blotting are far more
accurate and precise, and that these methods are clearly a better choice than
RT-PCR in most cases.
For example, one netter reported that by using RPA, he could detect
approximately 1x10^6 target transcripts, which he felt was sensitive enough.
In addition, it has been estimated that a northern blot is about tenfold less
sensitive than RPA, with minimum detection at approximately 1x10^7 target
transcripts. [6]
Another person said he would only be confident to conclude a result if an
experiment that was repeated three times by conventional northern and/or in
situ hybridization analysis matched that obtained by RT-PCR. Not only is
this a good idea for confirming data found through RT-PCR, but it is also
scientifically sound.
Unfortunately, because both RPA and northern blotting are more labor intensive
and require much more starting material (10-20 ug of RNA per lane), they are
limited to studying those genes that are relatively highly expressed, excluding
many interesting genes that might be expressed at much lower levels, or rare
pathological samples.
Most feel that PCR quantitation can and has been done poorly in many studies,
and that this, not the method itself, has given it a poor reputation.
If done carefully, RT-PCR has the potential to be one of the most powerful of
techniques. One day perhaps RT-PCR will become the method of choice, but for
all but a few exceptions, the old tried-and-true methods provide more solid
footing.
References
[1] Chelly, J. et al. (1990) Eur. J. Biochem. 187, 691-698
[2] Raeymaekers, L. (1993) Anal. Biochem. 214, 582-585
[3] Raeymaekers, L. (1995) Genome Res. 5, 91-94
[4] Celi, F. S., Zenilman, M. E. and Shuldiner, A. R. (1993) Nucl. Acids Res. 21, 1047
[5] Cottrez, F. et al. (1994) Nucl. Acids Res. 22, 2712
[6] Ferre', F. (1994) in The Polymerase Chain Reaction (Mullis, K. B., Ferre', F. and Gibbs, R. A., eds), p. 67, Birkhauser
Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health.
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You found this at the World Wide Web (WWW) Uniform Resource Locator (URL):
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/nov95.txt
Any reference to this column must be cited as the following published
article:
@article{Hengen1995Novtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Quantitative {PCR} - an
accurate measure of {mRNA}?",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "11",
pages = "476-477",
month = "November",
year = "1995"}
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******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * ******************************************************************************* |
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