Basic Principles of Quantitative PCR REVIEW 115

Basic Principles of Quantitative PCR

Luc Raeymaekers*

Abstract
The polymerase chain reaction (PCR) is an extremely sensitive method owing to the repetitive multipli-
cation of template molecules. This property is a drawback for quantitative measurements because small
differences in the multiplication factor lead to large differences in the amount of product. Two methods can
be used to solve the problem of quantification: kinetic methods based on the determination or comparison of
the amplification factor; and coamplification methods, which compare the amount of product to that of a
simultaneously amplified standard template. An overview of the theoretical background of both methods is
presented. For selection of a suitable method, both theoretical and practical considerations are important.
Kinetic methods are the most convenient if PCR can be performed without opening the tubes, as in some
apparatus using fluorescence detection. Coamplification methods can be done without expensive equip-
ment but requires the parallel running of several PCR tubes. When the number of initial template molecules
is close to one, as in the limiting dilution technique, statistical considerations become important.
Index Entries: PCR; quantitative; PCR, competitive; kinetics theory.

1. PCR Amplification
The polymerase chain reaction (PCR) is a re-
petitive amplification process by which in each
step (designated i), the copy number of product
(P) already accumulated during the previous step
(Pi - 1) is multiplied by a factor that depends on the
efficiency Ei of DNA synthesis during that step.
Ei is a measure of the relative increment of prod-
uct in one step, defined as
Ei = (Pi - Pi-1)/Pi-1 (1)
Because the copy number can at most double in
one step, Ei has a value between 0 and 1. The abso-
lute value of the increase of the copy number in
one cycle is
Pi - Pi-1 = Pi - 1 × Ei (2)
The amount of product accumulated after n cycles
is obtained by summation of the one-step incre-
ments:
*Author to whom all correspondence and reprint requests should be addressed: Laboratorium voor Fysiologie,
KULeuven, Campus Gasthuisberg O/N, B3000 Leuven, Belgium.
Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2000/15:2/115–122/$12.00
n
Pn = P 0 + ∑ P i-1 × Ei (3)
i=1
in which P0 denotes the starting amount of tem-
plate.
A mathematically equivalent expression for
Eq. 3 is the iterated product:
n
Pn = P 0 × ∏ (1 + E i). (4)
i=1
Because the values of Ei are not known a priori,
it follows from these equations that the absolute
amount of P0 cannot be determined from one
single measurement of Pn. However, the problem
of quantitative PCR can be easily solved (in prin-
ciple) in different ways. The procedures that have
been applied can be divided into two main catego-
ries: kinetic methods and coamplification meth-
ods. Another useful distinction that can be made is
between absolute quantification i.e., (the determi-

MOLECULAR B IOTECHNOLOGY 115 Volume 15, 2000

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nation of P0 in terms of number of molecules) and
relative quantification, (i.e., the measurement of
the ratio of P0 in various samples). Obviously, rela-
tive quantification requires less stringent controls
than absolute quantification.
2. Methods of Quantification
2.1. Kinetic Methods
If one makes the simplifying assumption that
the efficiency is constant in each cycle and tries
to approach this condition by limiting the number
of cycles, the equations given above can be re-
written such that the quantification of P0 becomes
possible. However, several theoretical (1,2) as
well as practical considerations suggest that the
fulfillment of stringent conditions should be met
before a constant efficiency can be safely as-
sumed.
By denoting the constant efficiency as E (with-
out subscript), Eq. 4 can be rewritten as:
Pn = P0 × (1 + E)n. (5)
It is possible to determine the value of E by
taking samples at several consecutive or noncon-
secutive cycles during the exponential phase of
the PCR, and by measuring the amount of prod-
uct Pi in each sample. The collection of more than
two samples is necessary (more is preferable) to
ascertain that the efficiency remains constant; in
other words, the PCR had not yet reached the
stage at which the efficiency starts to decrease. If
consecutive samples have been taken, E can be
determined from Eq. 1. Otherwise, the following
more general equation can be used, obtained by
rearranging Eq. 5 and replacing n by the param-
eter j (the number of cycles in the sampling inter-
val), Pn by Pj (the amount of product sampled at
the higher number of cycles), and P0 by Pi–j (the
amount of product sampled at the lower number
of cycles):
E = –1 + (Pi /Pi–j)1/j. (6)
Once the efficiency has been determined, P0 is
calculated from the measured amount of product
and the cycle number according to Eq. 7, which
is a rearrangement of Eq. 5:
MOLECULAR BIOTECHNOLOGY
Raeymaekers
P0 = Pi/(1 + E)i. (7)
P0 can be determined by plotting the logarithm
of the measured values of Pi as a function on n,
according to the logarithmic form of Eq. 5:
log Pn = log P0 + n × log (1 + E). (8)
The value of P0 can be read on the graph where
n equals zero. P0 also can be calculated by per-
forming a linear regression analysis of Eq. 8 (3).
It should be noted that for each of these proce-
dures, it is very important to obtain an accurate
value of E. Because of the exponential nature of
PCR, small differences in the value of E result in
appreciable differences in the amount of product.
For example, in two separate runs one condition
amplifying with an efficiency of one and in the
other one of 0.8 starting with identical copy num-
bers of template, the quantity of the resulting
products will differ by a factor of 24 after 30
cycles and by a factor of 68 after 40 cycles, respec-
tively. Another crucial consideration in this respect
is that for kinetic PCR, the method used to quan-
tify the products should give a signal that is linear
with the quantity of product, and that is not com-
pressed; when it is compressed, the degree of com-
pression should be small and accurately known.
Any compression of the signal that is not taken into
account results in an artifactual underestimation of
efficiency. Therefore, the construction of a stan-
dard curve based on a dilution series of the tem-
plate should be recommended for all quantitative
PCR applications, because it represents an addi-
tional control on the efficiency of amplification and
on the range of concentrations that can be quanti-
fied reliably. The methodology used in the ABI
PRISM™ 7700 system (Perkin Elmer, Foster City,
CA) and LightCycler (Roche Diagnostics, Basel,
Switzerland) is based on kinetic PCR in that the
apparatus continuously measures the amount of
product during the run (avoiding the complica-
tions of frequent opening and sampling of the
PCR tubes). The software of this system does not
extrapolate the amplification plot to the start of
the PCR, but instead calculates the threshold cycle
where the amplification plot crosses some signal
threshold. close resemblance of the intervening
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 Basic Principles of Quantitative PCR
2.2. Coamplification Methods
2.2.1. Principle of the Method
These methods quantify the sequence of inter-
est relative to a second control sequence by
coamplification in the same PCR tube. The con-
trol template can be a related cDNA whose con-
centration is precisely known and is amplified
using the same primer pair as for the unknown, or
it can be an unrelated cDNA corresponding to the
message of a constitutively expressed gene that is
used as a reference (for review, see 4-7). The main
advantages of this technique are that the results
are not affected by tube to tube variations in am-
plification efficiency, and it is not necessary to
restrict PCR to the exponential phase. Reliable
quantification is still possible if the PCR extends
into the linear phase or even into the saturation
phase, provided it is ascertained that the amplifi-
cation efficiency is the same for both templates
throughout the PCR, including the final cycles.
Quantitative coamplification rests on the assump-
tion that the product ratio of target and standard
sequences reliably reflects the ratio of their initial
copy numbers. Therefore, it is requisite that E is
identical for both sequences. In describing
coamplification PCR, we will make use of Eq. 4
given above, replacing the symbol P either by T
(the quantity of target sequence) or S (the quan-
tity of standard sequence).
n
T n = T 0 × ∏ (1 + E Ti )., (9)
i=1
n
(1 + E si ). (10)
Sn = S 0 × ∏
i=1
It is a prerequisite that the efficiencies for target
and standard are equal in each cycle, even as the
efficiencies decrease in the later cycles. There is a
good chance that this is indeed the case, because
the decrease of the rate of product synthesis with
the cycle number is determined mainly by a single
factor, namely the decrease of the ratio of bound
over free polymerase (1). If this requirement is ful-
filled ETi = E si for all values of i. It follows that in
these conditions when making the ratio of Eqs. 9
and 10, the iterated product terms cancel out, such
that the following equation is valid:
MOLECULAR B IOTECHNOLOGY
117
(Tn/Sn) = (T0/S0). (11)
Equation 11 allows one to calculate the un-
known initial copy number T0 from the known
quantities S0, Sn, and Tn:
T0 = (Tn × S0)/Sn. (12)
If the absolute value of S0 is not known, rela-
tive comparison of T0 with different samples is
still possible by adding the same quantity or de-
fined dilutions of S0 to each sample.
2.2.2. Coamplification
of the Target and an Unrelated Sequence
Reliable quantification by this method requires
stringent controls because the target and the stan-
dard sequences usually are unrelated, both with
respect to primer binding sites and the interven-
ing sequence. This situation increases the chance
that both templates are amplified with different
efficiencies, especially during the later linear
phase of PCR. Also, difficulty resides in the fact
that there is often a vast difference in the initial
copy number of both templates, usually the tem-
plate of the control gene (e.g., housekeeping gene)
being in excess. Without precautions, it is pos-
sible for PCR to saturate for the control sequence,
whereas the other one is still being amplified (see
also Subheading 3.1.). These difficulties do not
arise when using an engineered standard sequence
that resembles the target sequence, as described in
the next section. Amplifying the message of a con-
trol gene remains a useful and often-used proce-
dure to compare the amount of starting material
isolated from various samples in the experiment.
2.2.3. Coamplification
of the Target and a Closely Related Sequence:
Competitive PCR
A minimum requirement for reliable competi-
tive PCR is the identity of primer binding sites
allowing for the use of only one primer pair (how-
ever, some mismatches appear to be tolerated; see
8). To ensure equal amplification efficiency of
target and standard under all circumstances,
asequence (e.g. length, base composition) is rec-
ommended as well. Although quantification can
Volume 15, 2000
 118
Fig. 1 Idealized overview of a competitive PCR
experiment. A series of PCR tubes are spiked with the
same but unknown copy number (in the example 1
relative unit) of target sequence (T0) and with a dilu-
tion series in the example from 0.01 to 100 of a known
copy number of the standard sequence (S0). Schematic
gel patterns of the PCR products are shown, obtained
at the end of the exponential phase (Te and Se) and
after saturation of the PCR (T s and Ss). The graph
shows the standard curve constructed from the quanti-
fied gel bands. The value of T0 is equal to S0 at the
point of equivalence, i.e., where log (Tn/Sn) = 0.
be done by running a single PCR tube and apply-
ing Eq. 12, it is recommended to add an amount
of standard that does not differ too much from the
amount of target. In practice, reliable quantifica-
tion requires the analysis of several PCR tubes in
parallel, each containing the same T0 to be quanti-
fied but differing in the initial S0 added. The range
MOLECULAR BIOTECHNOLOGY
Raeymaekers
of the dilution series of S0 preferentially should en-
compass the copy number of T0 (9,10) except when
the copy number of T0 is so small that statistical
considerations become important; see Subheading
3.3.. The most convenient way to analyze the data
is to construct a standard curve by plotting the loga-
rithm of the product ratio of target and standard vs
the logarithm of the quantity of standard sequence
added to the tube (log S0) (10,11). From Eq. 11
one derives
log (Tn/Sn) = log T0 - log S0. (13)
It is clear from Eq. 13 that such a standard
curve should be a straight line with a slope of –1.
(Fig. 1) At the point of equivalence of Tn and Sn,
log (Tn/Sn) = 0 and log T0 = log S0. At this point
on the graph, the value of T0 to be determined
equals that of S0.
3. Sources of Error in Quantitative PCR
3.1. The Impact of the Plateau Phase on
Quantification
The exponential phase of the reaction extends
over a limited number of cycles because of the
accumulation of product. If several PCR tubes,
each containing a different initial amount of tem-
plate, are run in parallel, and if the amplification
is extended beyond the exponential phase into the
saturation phase, relative differences in the
amount of product will be smaller than the initial
differences. This is because tubes containing
more starting material will reach the saturation
phase sooner than tubes containing a smaller
amount. The phenomenon results in a systematic
bias against the more abundant PCR templates.
Therefore, relative quantifications between differ-
ent samples without coamplification of a resem-
bling standard sequence requires suitable controls
on the purely exponential nature of PCR in all
tubes to be compared. The same precautions ap-
ply to the method of coamplification of the se-
quence of interest with an unrelated,
constitutively expressed sequence (e.g., actin).
These housekeeping genes are often expressed at
much higher levels than the target sequence. The
product corresponding to such standard sequence
Volume 15, 2000
 Basic Principles of Quantitative PCR
may accumulate up to concentrations that inhibit
amplification, whereas the efficiency of amplifi-
cation of the target sequence is hardly diminished.
One of the advantages of competitive PCR, at
least in theory, is its insensitivity to the effect of
saturation of the PCR. However, an interference
of saturation with the quantification cannot be
fully excluded for some templates, as will be ex-
plained in the Subheading 3.2.
3.2. Standard Curves of Competitive PCR
Having a Slope Different from –1
In the original method of constructing a log–
log standard curve to evaluate competitive PCR,
the predicted property that the slope should equal
–1 has not been mentioned (10). As a conse-
quence many papers show standard curves that do
not conform to theory. Although this fact does not
necessarily imply that the quantifications based
on these curves are grossly wrong (see explana-
tions below), it is obvious that such errors should
be avoided in the future.
Because of the frequent occurrence of standard
curves with deviating slopes it is important to con-
sider possible causes. There are at least three
types of explanations:
1. If the PCR is run into saturation, a systematic
bias against the more abundant PCR products
may occur if their sequences differ signifi-
cantly (12). The consequence of this phenom-
enon is that the ratio of the products (Tn/Sn) is
smaller than the ratio of the initial copy num-
ber (T0/S0) when Tn is greater than Sn (or larger
when Tn is less than Sn). As a result, the slope
of the standard curve will be smaller than 1 in
absolute value. The position of the point of
equivalence is not shifted, so that the quanti-
fication based on the position of this point
is correct.
2. Systematic errors may arise in some methods
of quantification of the PCR products. It has
been observed that ethidium bromide-stained
bands yield a tilted standard curve when ana-
lyzed on agarose gels but not on polyacryla-
mide gels (13). Also in this situation, the point
of equivalence remains at the same position.
MOLECULAR B IOTECHNOLOGY
119
The phenomenon is probably a result of the
higher background staining in agarose gels.
3. A slope deviating from –1 may be caused by
the unequal amplification efficiencies of tar-
get and standard. The shift of the slope is ac-
companied by a shift of the point of
equivalence, resulting in erroneous quantifi-
cation. It can been shown by computer simu-
lation (14) that a deviation of the slope occurs
only if the difference between the amplifica-
tion efficiencies of target and standard varies
among PCR tubes of the dilution series on
which the standard curve is based. (A differ-
ence between ET and ES that is identical in all
tubes in all cycles results in a parallel shift of
the graph, thus maintaining the slope = 1
property but resulting in a shift of the point of
equivalence.) A possible cause is still specu-
lative. However, it is reasonable to suppose
that in some cases, two similar sequences that
amplify with the same efficiency during the
exponential phase may start to amplify with
different efficiencies during the later linear
stages of the PCR. Small differences in the
relevant properties of the templates may not
show up in conditions when DNA polymerase
and all substrates are in abundance, and the
concentration of the reaction products is still
below inhibiting levels. These differences may
become important, however, if the binding of
substrates or of polymerase, the rate of tem-
plate annealing, or the rate of strand dissocia-
tion become rate-limiting. Because the
different samples constituting the standard
curve contain different copy numbers of tem-
plate, each tube will spend a different number
of cycles in the nonexponential phase of the
PCR and will be differentially affected by the
difference between ET and ES.
3.3. Stochastic Effects in the Quantifica-
tion of Small Numbers of Molecules
It should be noted that the equations given in
the first part of this article are valid only if the
magnitude of influence of statistical variations on
the outcome of PCR can be neglected. Statistical
considerations become important when the num-
ber of template molecules is small and when the
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 120 Raeymaekers

efficiency is significantly smaller than 1, because
the amount of PCR product that is produced in
one cycle depends on molecular fluctuations. For
example, starting from a single copy of a DNA
sequence amplified with an efficiency of 0.8, the
probability that one copy remains after the first
cycle is 20%. In theory, the final copy number
after n cycles may be any number between 1 and
1.8n. As a consequence, the analytical equations
given above do not apply when the initial copy
number is low. A more rigorous description of the
PCR process in these conditions should be based
on the theory of branching processes; thorough
mathematical descriptions of PCR reactions in
these conditions have been published (15,16). The
simulation of PCR trajectories and the calcula-
tion of the expected outcome can be implemented
rapidly in suitable computer programs because
the distribution of Pi is the binomial distribution
with parameters Pn-1 and E (16)*. As expected
the confidence interval of the estimated initial
copy number of the target is larger for a low ini-
tial copy number. The uncertainty also increases
with decreasing amplification efficiency. For
instance, when the initial copy number is 100, the
relative uncertainty (ratio of uncertainty over true
value) is 0.1% for E = 0.9, and 25% for E = 0.5
(when the number of cycles is >20). The relative
uncertainty computed with one single initial copy
is 2.55 for E = 0.5, whereas it is 0.99 for E = 0.9.
Because the uncertainty increases with decreas-
ing initial copy number, it follows that the accu-
racy of coamplification PCR of a very low copy
number of target will be higher when using a
larger copy number of standard than by using simi-
lar copy numbers of standard and target (15). Sto-
chastic effects also may be important when PCR is
used in combination with the limiting dilution tech-
nique, as this method requires that many samples
contain one or a few template molecules (17,18). It
follows that the method is reliable only if the effi-
ciency equals or is very close to one.
3.4. Other Confusions
About Quantitative PCR
Besides concerns over the slope of competitive
PCR’s standard curve, the reader should be warned
of some other illegitimate—but nevertheless pub-
lished—simplifications in order to avoid a chain-
reaction multiplication of errors in the literature.
It has been stated that reliable quantification is
possible with competitive PCR, even when the ef-
ficiencies of the target (ET) and the standard (ES)
are different, assuming that the ratio ET/ES is a
constant value (19). This statement should be
made more precise in that it applies only to rela-
tive quantification and not to absolute quantifica-
tions, as can be seen from Eq. 14, which itself is
based on Eqs. 8 and 13:
log (Tn/Sn) = log (T0/S0) + n× log ([1 + ET]/[1 + ES]). (14)
Absolute quantification is possible only if the
term at the right of the plus sign n × log ([1 + ET]/[1
+ ES]) is equal to zero, i.e., if ET=ES. The deviation
of the quantification from the real value increases
with difference in amplification efficiency and with
number of cycles. In theory, relative quantification,
i.e., comparing T0 in different samples, is still pos-
sible as long as n and ET/ES remain constant. How-
ever, it seems that reliable relative quantification

The following is a simulation in the Mathcad program (version 6.0) of 500 PCR runs of 20 cycles, starting from one copy of
template that is amplified with an efficiency of 0.6 P denotes the copy number and m denotes the amplification factor (1+p), where p
is the probability of duplication, which for large copy numbers corresponds to the amplification efficiency. Rnd(1) generates a random
number between 0 and 1.
PROGRAM:
p=0.6 n=20 trials=500 j=1..trials P0j=1
i=1..n uijrnd(1) Pij=Pi–j+qbinom(uij,Pi-l,j,p) mij=Pij/Pi-l,j
RESULTS
Mean number of copies at 20 cycles Pn (standard deviation): 1.22 × 104 (5.984 × 103).
Mean amplification factor m (standard deviation):1.60 (0.007).
As described by Peccoud and Jacob (16), the mean value of m is an estimation of the real amplification factor that converges to the
real value as i tends to infinity. For a limited number of cycles, an estimation of P0 can be calculated for each run from the mean value
of mi according to Eq. 7. The value of P0 in this particular simulation was 1.01± 0.497 (mean ± standard deviation).

MOLECULAR BIOTECHNOLOGY Volume 15, 2000

 Basic Principles of Quantitative PCR
in these circumstances is possible only in theory,
as it would require too many controls to be feasible
in practice.
It has been stated that the ratio T0/S0 is propor-
tional to the ratio of the slope of the line relating
Tn to the number of cycles n, divided by the slope
of a similar graph for Sn (if both slopes are deter-
mined during the linear phase of the PCR, i.e.,
close to saturation; (20). There is neither a theo-
retical nor a practical reason why this should the
case. On the contrary, one would expect the
inverse because a sample containing more start-
ing material would run closer to saturation, and
consequently, show a less steep increase in the
amount of product as a function of n.
A simple PCR method for relative
quantitation has been proposed as an alternative
to other methods, such as competitive PCR (21).
The authors describe a method consisting of mak-
ing a series of progressive dilutions by mixing
the two samples to be compared in different ra-
tios. According to the authors, the alignment of
the quantities of amplified product in each tube
along a line would demonstrate that the ampli-
fication efficiency in each tube was equal, al-
lowing direct comparison between the two
samples. It is clear that this method does not
eliminate the trap of running the PCR close to or
into saturation, thereby compressing the differ-
ence between the amount of product in the two
samples. A close to linear regression line may be
obtained, even in conditions of near-saturation,
particularly if one allows for errors in the quan-
tification of the products. Furthermore, this pa-
per contains several mistakes in the calculations,
and it also shows graphs relating the amount of
PCR product to the number of cycles according
to which the amplification factor would be much
larger than 2, which is theoretically impossible.
Conclusions
The description of the PCR process shows that
the problem of making PCR quantitative in a reli-
able way is easily solved in theory. However, each
quantification requires several measurements, ei-
ther on several samples from one PCR tube (ki-
netic methods) or on several PCR tubes
MOLECULAR B IOTECHNOLOGY
121
(coamplification methods). The practical impli-
cations of this requirement should not be under-
estimated. When using a kinetic method,
frequent opening of the PCR tubes for sampling
during the run is inconvenient. Therefore, using
continuous florescent detection (as in the Perkin
Elmer ABI PRISM™ 7700) is an excellent solu-
tion, although the equipment is expensive.
Coamplification can be done with less invest-
ment, but each quantification requires several
PCR tubes, each of which must be further pro-
cessed for separate quantification of the target
and the standard. When doing competitive PCR,
perpetuation of the method needs especially
stringent precautions to avoid contamination of
samples and tools with the standard sequence, as
the latter is often handled from concentrated
stock solutions. In all cases, appropriate controls
should be done for each new sequence to be
quantified. Only then is it possible to obtain the
ideal combination of accurate quantification and
the extreme sensitivity offered by PCR.
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