overview of real-time PCR

key concepts of real-time PCR

1. Overview of
Real-Time PCR
Nucleic acid amplification and detection are among the most valuable techniques
used in biological research today. Scientists in all areas of research — basic
science, biotechnology, medicine, forensic science, diagnostics, and more —
rely on these methods for a wide range of applications. For some applications,
qualitative nucleic acid detection is sufficient. Other applications, however, demand
a quantitative analysis. Choosing the best method for your application requires a
broad knowledge of available technology.

This guide provides an introduction to many of the technical aspects of real-time

PCR. It includes guidelines for designing the best real-time PCR assay for your
experiments and explains how real-time PCR data are used in various applications.
In Sections 5–7, we present sample protocols and data that demonstrate the use
of real-time PCR in specific applications, namely, gene expression analysis, allelic
discrimination, and genetically modified organism (GMO) detection.

We hope that this guide will give you the information you need to bring this
powerful technique to your bench — and beyond.

1.1 Key Concepts of Real-Time PCR

1.1.1 What Is Real-Time PCR?
In conventional PCR, the amplified product, or amplicon, is detected by an
end-point analysis, by running DNA on an agarose gel after the reaction has
finished. In contrast, real-time PCR allows the accumulation of amplified product to
be detected and measured as the reaction progresses, that is, in “real time”.

Real-time detection of PCR products is made possible by including in the reaction

a fluorescent molecule that reports an increase in the amount of DNA with a
proportional increase in fluorescent signal. The fluorescent chemistries employed
for this purpose include DNA-binding dyes and fluorescently labeled sequence-
specific primers or probes. Specialized thermal cyclers equipped with fluorescence
detection modules are used to monitor the fluorescence as amplification occurs.
The measured fluorescence reflects the amount of amplified product in each cycle.

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 overview of real-time PCR
key concepts of real-time PCR

The main advantage of real-time PCR over conventional PCR is that real-time PCR
allows you to determine the starting template copy number with accuracy and
high sensitivity over a wide dynamic range. Real-time PCR results can either be
qualitative (presence or absence of a sequence) or quantitative (number of copies
of DNA). Real-time PCR that is quantitative is also known as qPCR. In contrast,
conventional PCR is at best semi-quantitative. Additionally, real-time PCR data can
be evaluated without gel electrophoresis, resulting in reduced experiment time and
increased throughput. Finally, because reactions are run and data are evaluated in
a closed-tube system, opportunities for contamination are reduced and the need
for postamplification manipulation is eliminated.

1.1.2 How Real-Time PCR Works

To understand how real-time PCR works, let’s start by examining a sample
amplification plot (Figure 1.1). In this plot, the PCR cycle number is shown on the
x-axis, and the fluorescence from the amplification reaction, which is proportional
to the amount of amplified product in the tube, is shown on the y-axis.

The amplification plot shows two phases, an exponential phase followed by a

nonexponential plateau phase. During the exponential phase, the amount of
PCR product approximately doubles in each cycle. As the reaction proceeds,
however, reaction components are consumed, and ultimately one or more of the
components becomes limiting. At this point, the reaction slows and enters the
plateau phase (cycles 28–40 in Figure 1.1).

Exponential phase Non-

0.3
exponential
plateau
phase
Fluorescence

0.2

CT value
0.1

Threshold line

0
0 10 20 30 40
Cycle

Fig. 1.1. Amplification plot. Baseline-subtracted fluorescence is shown.

© 2006 Bio-Rad Laboratories, Inc. All rights reserved. Real-Time PCR Applications Guide 3
overview of real-time PCR
key concepts of real-time PCR

Initially, fluorescence remains at background levels, and increases in fluorescence

are not detectable (cycles 1–18 in Figure 1.1) even though product accumulates
exponentially. Eventually, enough amplified product accumulates to yield a
detectable fluorescent signal. The cycle number at which this occurs is called the
threshold cycle, or CT. Since the CT value is measured in the exponential phase
when reagents are not limited, real-time qPCR can be used to reliably and
accurately calculate the initial amount of template present in the reaction.

The CT of a reaction is determined mainly by the amount of template present at

the start of the amplification reaction. If a large amount of template is present at
the start of the reaction, relatively few amplification cycles will be required to
accumulate enough product to give a fluorescent signal above background.
Thus, the reaction will have a low, or early, CT. In contrast, if a small amount of
template is present at the start of the reaction, more amplification cycles will be
required for the fluorescent signal to rise above background. Thus, the reaction will
have a high, or late, CT. This relationship forms the basis for the quantitative aspect
of real-time PCR. Details of how CT values are used to quantify a sample are
presented in Section 4.

1.1.3 Hallmarks of an Optimized qPCR Assay

Since real-time quantification is based on the relationship between initial template
amount and the CT value obtained during amplification, an optimal qPCR assay is
absolutely essential for accurate and reproducible quantification of your sample.
The hallmarks of an optimized qPCR assay are:

• Linear standard curve (R2 > 0.980 or r > |–0.990|)

• High amplification efficiency (90–105%)

• Consistency across replicate reactions

A powerful way to determine whether your qPCR assay is optimized is to run

serial dilutions of a template and use the results to generate a standard curve.
The template used for this purpose can be a target with known concentration
(e.g., nanograms of genomic DNA or copies of plasmid DNA) or a sample of
unknown quantity (e.g., cDNA). The standard curve is constructed by plotting
the log of the starting quantity of template (or the dilution factor, for unknown
quantities) against the CT value obtained during amplification of each dilution.
The equation of the linear regression line, along with Pearson’s correlation
coefficient (r) or the coefficient of determination (R2), can then be used to evaluate
whether your qPCR assay is optimized.

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 overview of real-time PCR
key concepts of real-time PCR

Ideally, the dilution series will produce amplification curves that are evenly spaced,
as shown in Figure 1.2 A. If perfect doubling occurs with each amplification cycle,
the spacing of the fluorescence curves will be determined by the equation 2n =
dilution factor, where n is the number of cycles between curves at the fluorescence
threshold (in other words, the difference between the CT values of the curves).
For example, with a 10-fold serial dilution of DNA, 2n = 10. Therefore, n = 3.32,
and the CT values should be separated by 3.32 cycles. Evenly spaced amplification
curves will produce a linear standard curve, as shown in Figure 1.2 B. The equation
and r value of the linear regression line are shown above the plot.

The r or R2 value of a standard curve represents how well the experimental data
fit the regression line, that is, how linear the data are. Linearity, in turn, gives a
measure of the variability across assay replicates and whether the amplification
efficiency is the same for different starting template copy numbers. A significant
difference in observed CT values between replicates will lower the r or R2 value.
You should strive for an r whose absolute value is >0.990 or an R2 value >0.980 for
your qPCR reactions.

Fig. 1.2. Generating a standard curve to assess reaction optimization. A standard curve was generated
using a 10-fold dilution of a template amplified on the iCycler iQ® real-time system. Each dilution was assayed in
triplicate. A, Amplification curves of the dilution series. B, Standard curve with the CT plotted against the log of
the starting quantity of template for each dilution. The equation for the regression line and the r value are shown
above the graph. The calculated amplification efficiency was 95.4%.

© 2006 Bio-Rad Laboratories, Inc. All rights reserved. Real-Time PCR Applications Guide 5
overview of real-time PCR
key concepts of real-time PCR

Amplification efficiency, E, is calculated from the slope of the standard curve using
the following formula:

E = 10–1/slope

Ideally, the amount of PCR product will perfectly double during each cycle of
exponential amplification; that is, there will be a 2-fold increase in the number of
copies with each cycle. This translates to a reaction efficiency of 2. Using an
efficiency equal to 2 in the equation above, 2 = 10–1/slope, indicates that the optimal
slope of the standard curve will be –3.32. Note that the absolute value of the slope
is the same as the ideal spacing of the fluorescent traces described above.

Amplification efficiency is also frequently presented as a percentage, that is,

the percent of template that was amplified in each cycle. To convert E into
a percentage:

% Efficiency = (E – 1) x 100%

For an ideal reaction, % Efficiency = (2 – 1) x 100% = 100%.

For the example shown in Figure 1.2:

E = 10–(1/–3.436) = 1.954

% Efficiency = (1.954 – 1) x 100% = 95.4%

At the end of each cycle, the amplicon copy number increased 1.954-fold,
or 95.4% of the template was amplified.
An efficiency close to 100% is the best indicator of a robust, reproducible assay.
In practice, you should strive for an amplification efficiency of 90–105%. Low
reaction efficiencies may be caused by poor primer design or by suboptimal
reaction conditions. Reaction efficiencies >100% may indicate pipetting error in
your serial dilutions or coamplification of nonspecific products, such as primer-
dimers. When using the method described above to determine amplification
efficiency, the presence of inhibitor can also result in an apparent increase in
efficiency. This is because samples with the highest concentration of template also
have the highest level of inhibitors, which cause a delayed CT, whereas samples
with lower template concentrations have lower levels of inhibitors, so the CT is
minimally delayed. As a result, the absolute value of the slope decreases and the
calculated efficiency appears to increase. If your reaction efficiency is <90% or
>105%, we suggest that you modify your assay by redesigning your primers and
probes. See Sections 2.3 and 2.4 for primer and probe design guidelines.

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