Quantitative PCR question

Dr. John Brunstein john.brunstein at helsinki.fi
Wed Jun 3 14:32:07 EST 1998

Hello Greg

	I've recently set up our lab for doing quantitative PCR, and after
reviewing all the available methods I chose what I think is both the
most laborious and the most accurate method: PLDA (PCR Limiting Dilution
Assay). The basic idea is simple, you set up serial dilutions of your
template and do replicate PCRs and score the frequency of + and -
reactions at each dilution. Rodrigo et. al (1) recently published a
paper describing a program QUALITY (and made the code available) for
calculation of the Poisson distribution function based on this data;
this allows both a quantitation and astandard error estimate to be made
from this data. (See also Taswell(2) for the underlying math).  It would
appear to me that previous authors using PLDA have based their
calculations on the assumption that for PLDA to work, it must be capable
of single-copy detection; however I believe this is erroneous... if your
detection limit is for instance 10 copies, your quantitation results are
still valid for comparing samples while no longer giving absolute values
(ie you can do relative but not absolute quantitation). In my opinion
ALL other PCR quantitation techniques including the new real-time
monitoring systems really only give you relative values as well. Other
people may (and I sure will) disagree as to whether PLDA is the 'best'
technique; for what I thought was a good discussion of the relative
merits of available techniques see Schäfer and Laufs (3).
	We've just gotten one of the PE/ABI 7700 real-time fluoresence based
PCR quantitation machines here, and while it all looks very spiffy (and
it's certainly easier and faster than PLDA), it's NOT cheap (either to
buy the machine, or to have ABI make the reporter probes..which have to
be custom synthesized for each target by ABI, as I understand it they
are not licensing the chemistry out). Also, it can't be used in a
multiplex manner, whereas PLDA can allowing for simultaneous
quantitation of a standard or marker with your target.
	As the saying goes, the above opinions are freely given and worth what
you paid for them.

(1) Rodrigo A, P. Goracke, K. Rowhanian, and J. Mullins. Quantitation of
Target Molecules from Plymerase Chain Reaction-Based Limiting Dilution
Assays. Aids Res. and Human Retroviruses 13(9):737-742 (1997)
	see also http://ubik.microbiol.washington.edu/cbu/quality

(2) Taswell, C. Limiting Dilution Assays for the Determiniation of
Immunocompetent Cell Frequencies. J. Immunol. 126(4):1614-1619 (1981)

(3) Schäfer P, and R. Laufs. Experience with Quantitative PCR for the
Management of HCMV Disease. Intervirology 39:204-212 (1996)
*   Dr. John Brunstein                    *
*   Dept. of Virology                     *
*   Haartman Institute                    *
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