DNA/RNA Real-Time Quantitative PCR
DNA/RNA Real-Time Quantitative PCR
The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. As little as a single copy
of a particular sequence can be specifically amplified and detected. Theoretically, there is a quantitative relationship
between amount of starting target sequence and amount of PCR product at any given cycle. In practice, though, it is
a common experience for replicate reactions to yield different amounts of PCR product. The development of real-time
quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the
routine and reliable quantitation of PCR products.
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Flourogenic 5' Nuclease Chemistry
Two fluorescent dyes, a reporter (R) When both dyes are attached to During each extension cycle, the Once separated from the quencher,
and a quencher (Q), are attached to the probe, reporter dye emission Taq DNA polymerase cleaves the the reporter dye emits its characteristic
the 5' and 3' ends of a TaqMan® probe. is quenched. reporter dye from the probe. fluorescence.
Figure 1. Stepwise representation of the forklike-structure–dependent, polymerization-associated, 5' to 3' nuclease activity of Taq DNA
polymerase acting on a fluorogenic probe during one extension phase of PCR.7
they can be labeled with different, distinguishable reporter dyes. By using probes labeled with different reporters,
amplification of two distinct sequences can be detected in a single PCR reaction. The disadvantage of fluorogenic
probes is that different probes must be synthesized to detect different sequences.
Instrumentation
PE Biosystems has two instruments designed to detect fluorescence during the thermal cycling of PCR. The
simpler system is the GeneAmp® 5700 Sequence Detection System. This complete system consists of an Optical
Detector and a GeneAmp® PCR System 9600, coordinately controlled by software running on a Windows®-based
computer. The system has been designed for efficient detection of PCR product accumulation using either SYBR®
Green I double stranded DNA binding dye or TaqMan® fluorogenic probes. The 96 reaction tubes are irradiated
with a white light source and the resulting fluorescence is detected using a CCD array to capture an image of all
96 wells. The software collects the images throughout the thermal cycling of PCR and analyzes the data to generate
an amplification plot for each reaction. Fluorogenic probes labeled with fluoroscein can be detected on the 5700
system, but the instrument does not have the capability of distinguishing two or more fluorophores. Despite this
single-color detection limitation, however, the 5700 system is still able to use an internal reference dye (ROX) to
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normalize for non-PCR-related, well-to-well fluctuations in fluorescence. This ability to normalize is achieved by
using fluorescence readings taken at 95 ºC in the baseline region and is essential for reproducible results.
The ABI PRISM® 7700 Sequence Detection System is a more flexible system designed to take full advantage of the
benefits of fluorogenic probe detection. The 7700 system has a built-in thermal cycler and a laser directed via fiber
optic cables to each of the 96 sample wells. The fluorescence emission travels back through the cables to a CCD
camera detector. Because each well is irradiated sequentially, the dimensions of the CCD array can be used for
spectral resolution of the fluorescent light. This contrasts with the 5700 system, in which the CCD is used for spatial
resolution of the 96 wells. Because the 7700 instrument detects an entire fluorescence spectrum, the system is
capable of distinguishing and quantitating multiple fluorophores in each sample well. The software analyzes the
data by first calculating the contribution of each component dye to the experimental spectrum. Each reporter signal
is then divided by the fluorescence of an internal reference dye (ROX) in order to normalize for non-PCR related
fluorescence fluctuations occurring well-to-well or over time. The use of this internal reference dye, enabled by the
ability to distinguish fluorophores, increases the precision of the data obtained with the 7700 system. The fluores-
cence emissions of SYBR® Green I dye and ROX dye are well resolved, so the benefit of using an internal reference
dye is obtained for SYBR® Green I dye detection of PCR on the 7700 system. The other advantage of distinguish-
ing fluorophores is that probes labeled with different reporter dyes can be used so that more than one PCR target can be
detected in a single tube.
Figure 2. Model of a single amplification plot, showing terms commonly used in real-time quantitative PCR.
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3a) Rn vs Cycles
4a)
16
10 1.2
Fluorescence (∆nR)
8
1.0
Rn 6
0.8
4
0.6
2
0.4
0
0.2
-2
0.0
-4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 0 5 10 15 20 25 30 35 40
Cycle Number Cycle Number
3b) Rn vs Cycles
100 4b)
Variable PCR Plateau
96 replicates
0.10
10
0.08
20 21 22 23 24 25 26 27
0.04
20 21 22 23 24 25 26 27 2828
1
0.02
0.00
0.1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
16
-0.4 0 0.4 0.8 1.2 1.6 2 2.4 2.8
Log CO
Figure 3a shows amplification, using the 5700 system, of the human β-actin gene in five-fold dilutions of genomic
DNA. In this figure, the change in fluorescence of SYBR® Green I dye is plotted versus cycle number. Six replicates
were run for each DNA amount. Figure 3b shows the same data, but with the log of the change in fluorescence
plotted versus cycle number. The 5700 system software calculated the CT (threshold cycle) for each reaction.
The CT values are plotted versus the log of the initial amount of genomic DNA to give the standard curve shown
in Figure 3c.
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These three plots illustrate the basic principles of real-time PCR quantitation. The higher the initial amount of
genomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the CT value. CT values
are very reproducible in replicates because the threshold is picked to be in the exponential phase of the PCR. This is
shown in Figure 3b where the threshold intersects the amplification plots in the region where there is a linear relation
between log of the change in fluorescence and cycle number. In the exponential phase, reaction components are not
limiting and replicate reactions exhibit uniform and reproducible results.
Advantages of Real-Time
The development of competitive PCR was driven by a reliance on endpoint measurements. Determining CT values
by following the real-time kinetics of PCR eliminates the need for a competitor to be co-amplified with the target.
Quantitation can be performed by the more basic method of preparing a standard curve and determining unknown
amount by comparison to the standard curve. Compared to endpoint measurements, the use of CT values also
expands the dynamic range of quantitation because data is collected for every cycle of PCR. A linear relationship
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between CT and initial DNA amount has been demonstrated for over five orders of magnitude, compared to the one
or two orders of magnitude typically observed with an endpoint assay. Real-time quantitation eliminates post-PCR
processing of PCR products, which not only increases throughput and reduces the chances for carryover contamina-
tion, but also removes post-PCR processing as a potential source of error. Although not immune, CT values are less
sensitive than endpoint values to the effects of PCR inhibitors, again, because measurements are from the exponen-
tial phase where reaction components are not limiting.
10000
1000
Relative Quantity
100
10
2 hr
6 hr
overnight
®
7700/TaqMan Assay
IL-2
untreated
2 hr
6 hr
overnight
untreated
2 hr
TNF-α
6 hr
overnight
Figure 5. Relative quantitation results for three cytokine mRNAs in PBMCs that have been stimulated with CD3/CD28 antibodies. (Data
generated in collaboration with DNAX Research Institute.)
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The results shown in Figure 5 were normalized using the CT values obtained for the ribosomal RNA amplifica-
tions run in the same plate. The 18S ribosomal RNA is an endogenous control that is used to normalize the samples
for differences in the amount of total RNA added to each cDNA reaction. The use of this endogenous reference also
normalizes for variation in reverse transcriptase (RT) efficiency among the different cDNA reactions. Variation in
RT efficiency other than sample-to-sample variation is controlled for because a single cDNA reaction is performed
for each total RNA sample. This single cDNA reaction is then split to perform the target and control amplifications.
At high levels of RNA, Figure 5 shows that approximately the same relative quantitation results are obtained
whether SYBR® Green I dye detection or fluorogenic probe detection is used on either the 5700 or 7700 system.
Thus, at the 2 hr time point, the increase in cytokine mRNA level is approximately 2000-fold for IL-2, 100-fold for
IL-4, and 40-fold for TNF-α, regardless of analysis method used. At low levels of RNA, though, the detection of
non-specific amplification by SYBR® Green I dye complicates the results. For the IL-2 analysis in the overnight
sample, fluorogenic probe detection on either the 5700 or 7700 system shows the level of IL-2 mRNA is about
0.75× the amount in the untreated sample. Using SYBR® Green I dye detection, the amount of IL-2 mRNA in the
overnight sample appears to be 3.5×. This inflated value observed with SYBR® Green I dye detection is due to the
detection of non-specific amplification products. The TNF-α levels observed in the overnight sample reveal a
different consequence of non-specific amplification. Both analyses using fluorogenic probe detection indicate the
TNF-α level is about 0.6× compared to the untreated sample. The two SYBR® Green I dye results give wildly
different values of 1× and 0.03×. Non-specific amplification is not necessarily consistent well-to-well, so replicates
can give much different results. Thus, non-specific amplification can lead to erroneous and/or highly variable results
at low target levels when SYBR® Green I dye or another generic DNA-binding dye is used for detection. For these
particular cases, lower primer concentrations were tried to increase the specificity of the amplifications. Changing
primer concentrations from 900 nM to 50 nM reduced the spurious results observed with SYBR® Green I dye detection.
The use of fluorogenic probes avoids the complications caused by detection of non-specific amplification. Because
non-specific amplification is more of a problem at low target levels, fluorogenic probe assays tend to be more
sensitive for detection of low amounts of target and have a greater dynamic range compared to assays using DNA
binding dyes. Another advantage of fluorogenic probes is that, on the ABI PRISM® 7700 system, the target and endog-
enous control (e.g., rRNA) amplification can be performed in the same tube. This is possible because target and
control probes can be labeled with distinguishable reporter dyes. This reduces the number of reactions that need to
be run and ensures that exactly the same amount of template is available for target and control amplification. Also, the
inclusion of an in-tube internal positive control increases confidence in the results obtained for target quantitation.
Detection on the ABI PRISM® 7700 system tends to give lower coefficients of variation than detection on the
GeneAmp® 5700 system. Improved precision means that smaller differences in initial copy number can be distinguished.
Conclusions
Compared to endpoint quantitation methods, real-time PCR offers streamlined assay development, reproducible
results, and a large dynamic range. Real-time PCR eliminates the need for competitive in-tube standards with
identical primer sets as targets. Thus, the process of creating quantitative assays is streamlined because the con-
struction and characterization of such standards are no longer required. Real-time PCR now makes quantitation
of DNA and RNA much more precise and reproducible because it relies on CT values determined during the expo-
nential phase of PCR rather than endpoint. In addition, the use of CT values allows a larger dynamic range. This
increases throughput because it is no longer necessary to analyze dilutions of each sample in order to obtain accu-
rate results.
The researcher now has a number of options for implementing real-time quantitation in his or her lab. Homoge-
neous detection of PCR products can be done using double-stranded DNA binding dyes or fluorogenic probes.
Detection of fluorescence during the thermal cycling process can be performed using either the GeneAmp® 5700
or ABI PRISM® 7700 Sequence Detection Systems. Choosing among these options requires balancing the demands
of sensitivity, convenience, precision, and cost.
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