Chapter 8
Multiplex Analyses Using Real-Time Quantitative PCR
Steve F.C. Hawkins and Paul C. Guest
Abstract
Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each
probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The
signal from each dye is used to quantitate the amount of each target separately in the same tube or well.
The availability to multiplex therefore allows the measurement of the expression levels of several targets or
genes of interest quickly. Here, we describe a method using the SensiFAST and SensiFAST One-Step
probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.
Key words qPCR, Fluorescent dyes, Taq polymerase, Quantitation, mRNA, cDNA, Amplicon,
Multiplex analysis
1
Introduction
Polymerase chain reaction (PCR) method was a revolutionary
innovation by Kary Mullis in the 1980s [1, 2]. Since this time, it
has seen widespread use in biomedical research since it can detect
and quantify small amounts of specific nucleic acid sequences. For
example, small levels of messenger RNA (mRNA) can be quantified through the combination of reverse transcription (RT) to yield
complementary DNA (cDNA) and PCR amplification to produce
exponentially higher levels of these cDNA strands [3] (Fig. 1). In
addition to increased levels of the amplified products (amplicons),
the reliability and reproducibility of measurements between different laboratories are essential, especially if the method is to be performed in a clinical setting. This is critical for patient outcomes as
well as for reducing healthcare costs since approximately one third
of medical care budgets result from measurements and tests associated with diagnosis [4].
Quantitative PCR (qPCR) is a later development of the method
that allows users to monitor the progress of a PCR reaction in real
time [5]. In brief, the method uses a DNA-based sequence-specific
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546,
DOI 10.1007/978-1-4939-6730-8_8, © Springer Science+Business Media LLC 2017
125
+
cDNA
Fig. 1 Schematic diagram of PCR
Nucleotides
3’
5’
+
Primer
5’
3’
3’
Denaturation
5’
5’
3’
Annealing
3’
5’
5’
3’
Elongation
3’
5’
5’
3’
Repeat
20-40 cycles
Repeat
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Steve F.C. Hawkins and Paul C. Guest
Quantitative PCR
127
probe with a fluorescent reporter molecule at one end and a molecule that quenches this fluorescence at the other. The proximity
of the reporter to the quench molecule prevents the detection of
fluorescence and cleavage of the probe by the 5′ to 3′ exonuclease
activity of Taq polymerase results in unquenched emission of fluorescence. Thus, the increase in the cDNA amplicon targeted by the
reporter probe during each PCR cycle leads to a proportional
increase in fluorescence due to cleavage of the probe and release of
the reporter (Fig. 2). The available fluorescent reporter molecules
include dyes that bind to double-stranded DNA such as SYBR®
Green (Thermo Fisher Scientific; Waltham, MA, USA) or sequence
specific probes like Molecular Beacons (Newark, NJ, USA),
Scorpions (DxS Ltd), or TaqMan® Probes (Roche Molecular
Diagnostics; Basel, Switzerland). As with standard PCR, qPCR is
normally performed using a thermal cycler, which can rapidly heat
and cool samples to allow the melting, annealing, and extension
phases of replication. However in the case of qPCR, the thermocycler should also have the ability to illuminate each sample with
specific wavelengths of light for the detection of the fluorescence
emitted following excitation of the probe.
PCR normally consists of a series of temperature changes that
are repeated approximately 30 times. Each cycle consists of two or
three steps. In the three step cycling approach, the first step is carried out at approximately 95 °C, which allows separation of the
double-stranded nucleic acid chains (denaturation). The second
phase is performed at around 55 °C to allow binding of the primers to the DNA/cDNA template (annealing). Finally, the third
step is carried out at 72 °C to facilitate polymerization using DNA
polymerase (elongation). In the two step cycling method, the
Reporter
Quencher
5’
5’
3’
Probe
3’
Primer Nucleotides
cDNA
3’
5’
Denaturation
3’
Taq polymerase cleaves
reporter yielding unquenched
fluorescent signal
5’
5’
Annealing
3’
5’
3’
Repeat
~2 x 1020-40
reporter
molecules
Elongation
20-40 cycles
Repeat
3’
Fig. 2 Schematic diagram of real-time qPCR procedure
5’
3’
5’
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Steve F.C. Hawkins and Paul C. Guest
annealing and elongation steps are combined at the temperature
annealing temperature. In qPCR, it should be noted that 40 cycles
are performed and that the temperatures and associated times used
in each cycle depend on a variety of factors, such as the polymerase
used, the concentration of deoxyribonucleotides (dNTPs), and the
optimum binding temperature of the primers.
In general, two basic methods are used in qPCR and these are
based on ether relative quantification and absolute quantification
[6]. Relative quantification is based on comparisons with standard
DNA/cDNAs within the sample for measurement of ratiometric
differences. The absolute quantitation approach can yield the precise number of resulting amplicons by comparison with DNA standards using a calibration curve. This requires that PCR of the
DNA/cDNA in the sample and the standard have the same amplification efficiency. In addition to widespread use in research studies, qPCR has already been applied in many studies for the discovery
of biomarkers for applications in clinical studies such as evaluating
the status of certain cancers or for monitoring disease progression
or treatment response [7–9]. Here, we describe the use of the
SensiFAST™ Probe (Bioline; London, UK) that uses a unique buffer chemistry to enable fast and reproducible multiplex qPCR
determinations. This property makes this an ideal approach for
routine clinical use.
2
Materials (See Note 1)
1. 400 nM oligonucleotide primers (see Notes 2 and 3).
2. 100 nm probes (see Notes 4 and 5).
3. Templates: approximately 1 mg genomic DNA or 100 ng
cDNA or 1 × 10−6–1.0 μg total RNA or 0.01 pg mRNA (see
Note 6).
4. 1× SensiFAST Probe Mix, containing hot-start DNA polymerase, dNTPs, stabilizers, and enhancers.
5. Reverse transcriptase (see Note 7).
6. RNase inhibitor (see Note 7).
7. qPCR thermocycler (see Note 8).
3
Methods (See Note 9)
1. Isolate DNA or RNA as required using standard methods.
2. Select amplicons of interest (see Note 10).
3. For DNA and cDNA templates, prepare a PCR master mix based
on a standard 20 μL final reaction volume containing the primers, probes, template, and probe mix (see Notes 6 and 11).
Quantitative PCR
129
0.44
0.40
0.36
Fluorescence
0.32
0.28
0.24
0.20
0.16
0.12
0.08
0.04
0
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 41 42 43 44 45
Cycle
Fig. 3 Five replicates were run using a conventional TaqMan primer/probe set under fast cycling conditions
(3 min 95 °C followed by 45 cycles at 95 °C for 10 s and 60 °C for 10 s). Singleplex reactions (blue line) and
quadruplex reaction (red line) for the γ-actin and JOE dye are indistinguishable. However, slightly lower fluorescence intensity is often seen for the multiplex reactions, as reagents are consumed more quickly
4. For total RNA and mRNA templates, prepare a PCR master
mix based on a standard 20 μL final reaction volume containing the 1:100 reverse transcriptase, 1:50 RiboSafe RNase
Inhibitor, primers, probes, template, and probe mix (see Notes
6 and 11).
5. Suggested thermal cycling conditions for DNA and cDNA: 1
cycle at 95 °C for 2–5 min for polymerase activation, then 40
cycles at 95 °C for 10 s for denaturation and 60 °C for 20–50 s
for annealing/extension (see Notes 12 and 13) (Fig. 3).
6. Suggested thermal cycling conditions for RNA: 1 cycle at
45 °C for 10 min for reverse transcription, 1 cycle at 95 °C for
2 min for polymerase activation, then 40 cycles at 95 °C for 5 s
for denaturation and 60 °C for 20 s for annealing/extension
(see Notes 13 and 14).
7. Data analysis (see Note 15).
4
Notes
1. These guidelines refer to the design and setup of TaqMan
probe-based PCR. Please refer to the relevant literature when
using other probe types. The specific amplification, yield, and
overall efficiency of any qPCR can be critically affected by the
sequence and concentration of the probes and primers, and
amplicon length.
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Steve F.C. Hawkins and Paul C. Guest
2. Use primer-design software, such as Primer3 (http://frodo.
wi.mit.edu/primer3/) or visual OMP™ (http://dnasoftware.
com/). Primers should have a melting temperature (Tm) of
approximately 60 °C; the Tm of the probe should be approximately 10 °C higher than that of the primers. Tm can be determined using software; however, a good approximation is
(2 × number of As and Ts) + (4 × number of Gs and Cs).
Importantly, a 40–60 % GC content is recommended for all
primers and to avoid long stretches of any one base. There is a
range of about 6–8 °C over which the PCR will work well. The
closer you are to the top of this range, the more specificity you
will have. For fast reaction kits such as the SensiFAST we also
recommend adding a further 5 °C as they have a higher salt
concentration.
3. A final primer concentration of 400 nM is suitable for most
probe-based reactions; however to determine the optimal concentration we recommend titrating in the range 200–
1000 nM. The forward and reverse primers concentration
should be equimolar. We recommend aliquoting the primers
to avoid repeated freeze/thaw of the primary source, as this
will effect PCR efficiency and sensitivity. Aliquots should be
used for up to six freeze/thaw cycles.
4. A probe concentration of 100 nM is recommended for multiplexing since higher concentrations can result in cross-channel
fluorescence. Probe sequence should be designed as above.
5. For each probe, consider the spectral properties of the dyes in
terms of intensity of fluorescence and spectral overlap with
other dyes within the reaction. It is important to determine the
dyes for which your qPCR instrument has been calibrated or is
capable of detecting once calibrated. The manufacturer can
provide instrument excitation and detectable emission wavelengths (Table 1). Some of the older instruments require the
use of a passive reference such as ROX or fluorescein, to normalize expression levels between wells of a 96 or 384-well
plate. If normalization is required with these instruments, this
will reduce the selection of fluorescent dyes that can be used.
6. It is important that the DNA template is suitable in terms of
purity and concentration. The template must be devoid of any
contaminating PCR inhibitors (e.g., EDTA). The recommended amount of template for PCR is dependent upon the
type of DNA used. For genomic DNA, use up to 1 mg extracted
DNA using a kit such as the Bioline ISOLATE II Genomic
DNA Kit or a phenol/chloroform-based method such as
Bioline TriSure (ensure that samples are washed thoroughly as
even small amounts of phenol are inhibitory to PCR). For
cDNA, highly pure RNA is recommended and to perform a
two-step RT-PCR. It is also important to use a pre-optimized
Quantitative PCR
131
Table 1
Common fluorophores and quenchers used for qPCR probes
Fluorophore
Absorption (nm)
Emission (nm)
Suggested compatible quencher
FAM
495
517
TAMRA, BHQ-1, Dabcyl
JOE
520
548
TAMRA, BHQ-1, Dabcyl
VIC
528
546
TAMRA, BHQ-1, Dabcyl
HEX
537
553
TAMRA, BHQ-1, Dabcyl
NED
546
575
TAMRA, BHQ-1, Dabcyl
TAMRA
550
576
BHQ-2
Cy3
550
570
BHQ-2
ROX
581
607
BHQ-2
Cy5
650
667
BHQ-2/BHQ-3
mix such as the SensiFAST cDNA Synthesis Kit for reverse
transcription. The optimal amount of cDNA to use in a single
PCR is dependent upon the copy number of the target gene.
We suggest using 100 ng cDNA per reaction; however, it may
be necessary to vary this amount. For RNA, it is important that
the template is intact and devoid of DNA or contaminating
inhibitors of both reverse transcription and PCR. The recommended amount of template for one-step real-time RT-PCR is
dependent upon the type of RNA used. For total RNA, we
recommend using 1 × 10−6 pg to 1 μg and for mRNA 0.01 pg
per 20 μL reaction.
7. For use with RNA templates. It is important to use and RNAase
inhibitor such as the RiboSafe RNase inhibitor (Bioline)
although others can be used.
8. Many instruments can be used here such as the 7500 FAST,
7900HT FAST, ViiA7™, and StepOne™ from Applied
Biosystems (Waltham, Massachusetts, USA), the Mx4000™
from Stratagene/Agilent (Santa Clara, California, USA), the
iCycler™ and MyiQ5™ from Bio-Rad (Hercules, California,
USA), the LightCycler® from Roche (Basel, Switzerland), the
RotorGene™ from Qiagen (Hilden, Germany), and the MIC
from Bio Molecular Systems (Upper Coomera, Queensland,
Australia). Although other instruments can be used but it is
important to check with the manufacturer for compatibility, as
described above.
9. qPCR is extremely sensitive and so to help prevent any carryover DNA contamination, separate areas for reaction setup,
PCR amplification and any post-PCR gel analysis should be
maintained. It is essential that any tubes containing amplified
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Steve F.C. Hawkins and Paul C. Guest
PCR product are not opened in the PCR setup area. As with all
types of PCR, follow the three-room rule. One of the biggest
causes of contamination and background is from using the
same pipettes for extraction, PCR setup, and post-run analysis.
Even if aerosol resistant tips are used all the time, this is not a
good idea. Instead, you should have a dedicated set of pipettes
for each stage. In addition to pipettes, you should have a different location, either hoods with UV lamps (or preferably a
completely different room) for extractions, PCR setup, and
any post PCR analysis. In addition, it is important to detect the
presence of contaminating DNA that may affect the reliability
of the data by including a no-template control reaction, replacing the template with PCR-grade water.
10. For multiplex analyses, the length of the amplicons should be
similar and between 50 and 150 bp for optimal PCR efficiency.
This is important since each amplicon competes for the same
reagents in the probe mix (dNTPs and polymerase).
11. Always mix the reagents well before use. This may sound obvious but this is a very sensitive system and the reagents contain
dyes, dNTPs, and enzymes that may have settled while sitting
in the freezer or refrigerator.
12. The conditions are suitable for the SensiFAST Probe Kit targeting amplicons up to 200 bp. For the polymerase activation
step, 2 min are required for cDNA and 5 min for genomic
DNA. For all other steps, the temperatures may vary depending on the primer sequences and up to 50 cycles may be
required in multiplex experiments.
13. When testing a mix, template, or primers, it is important to
amplify from a tenfold template dilution series. Loss of detection at low template concentrations is the only direct measurement of sensitivity and can also indicate the presence of
inhibitors. If inhibition is observed, either the DNA needs to
be used at lower concentrations or it requires re-purification.
Ideally, samples should cross the threshold (Ct) between cycles
20–30. Therefore, individual reactions should be optimized
prior to multiplexing, with efficiencies as close to 100 % as
possible.
14. The conditions are suitable for the SensiFAST Probe One-Step
Kit, targetting amplicons of up to 200 bp. However, they can
be varied to suit different machine-specific protocols. The
reverse transcription reaction time can be extended up to
20 min and/or the temperature can be increased up to
48 °C. For the annealing/extension stage, temperatures may
vary depending on primer sequences and up to 50 s may be
necessary for multiplexing with more than two probes.
Quantitative PCR
133
15. Optimal analysis settings, such as baseline and threshold values, for each primer/probe set are a prerequisite for accurate
quantification data. It is therefore important to analyze the
data for each channel separately as the qPCR instrument
default settings may not provide accurate results. It is recommended to keep the multiplex reactions after amplification so
that if there is any doubt in the results, the PCR products can
be checked on an agarose gel.
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