qPCR Technical Guide

Detection Methods

Primer and Probe Design

Instrumentation

Applications Guide
 Table of Contents
Introduction.............................................................................. 1 Optimizing qPCR.................................................................... 18
Guidelines for Optimizing Both qPCR and qRT-PCR................ 18
Quantitative PCR: How does it work?.................................... 2
Check Primer Design for Primer-Dimer Potential................ 18
qPCR Detection Methods......................................................... 3 Optimize Primer Concentrations........................................ 18
Dye-Based Detection............................................................... 3
qPCR Technical Guide

Optimize Probe Concentration.......................................... 19

DNA Binding Dyes – How They Work.................................. 3 Validate Performance with a Standard Curve..................... 20
Melt/Dissociation Curves..................................................... 3 Prepare a Melt Curve......................................................... 21
Advantages and Disadvantages of Dye-Based Detection...... 4 Set the Threshold Value..................................................... 21
Probe-Based Detection............................................................ 5 Additional Guidelines for Quantitative Reverse
Primer and Probe Design......................................................... 6 Transcription PCR (qRT-PCR).................................................. 22
Linear Probes.......................................................................... 6 Verify RNA Quality............................................................. 22
Hydrolysis Probes Confirm that Primers Span or Flank Long Introns............... 22
(Dual Labeled Fluorescent Probes or TaqMan)...................... 6 Conduct No-Reverse Transcriptase (no-RT) Controls........... 23
Hybridization Probes............................................................ 6 Optimize Reverse Transcription.......................................... 24
Structured Probes.................................................................... 7 Additional Optimization for Multiplex Reactions.................... 26
Molecular Beacons Probes................................................... 7 Check Primer Design......................................................... 26
Scorpions Probes................................................................. 7 Optimize Primer Concentrations........................................ 26
Modifications.......................................................................... 8 Optimize Mg2+ Concentration........................................... 26
Locked Nucleic Acid............................................................ 8 qPCR Reagent Selection Table............................................... 28
Minor Groove Binders......................................................... 9
Troubleshooting..................................................................... 29
Template, Primer Design, Probe Design,
Fluorescence Issues............................................................... 29
Dye Choice and Quenchers..................................................... 9
Dissociation/Melting Curves.................................................. 30
Template Considerations..................................................... 9
Standard Curve..................................................................... 31
Primer Design Considerations.............................................. 9
qRT-PCR Specific................................................................... 33
Probe Design Considerations............................................. 10
Multiplex............................................................................... 33
Dye Choice in Probe Design............................................... 10
Product Specific.................................................................... 33
Quenchers in Probe Design................................................ 10
Primer and Probe Design Software and Web Sites.............. 11 Appendix 1: Traits of Commonly Used Fluorophores.......... 34
Instrumentation..................................................................... 12 Appendix 2: How to Optimize Your
Considerations...................................................................... 12 Quantitative Real-Time RT-PCR............................................. 35
Components..................................................................... 12 Introduction.......................................................................... 35
Sensitivity.......................................................................... 12 Tissue Sampling and RNA Extraction..................................... 35
Specificity of Detection...................................................... 12 RNA Quantity and Integrity................................................... 35
Dynamic Range................................................................. 12 Reverse Transcription............................................................. 36
Detection Linearity............................................................ 12 Elevated Fluorescence Acquisition......................................... 36
Software........................................................................... 12 Crossing Point Data Evaluation.............................................. 37
High-Throughput qPCR Instruments...................................... 13
References.............................................................................. 38
Applied Biosystems (ABI)................................................... 13
Roche Applied Science...................................................... 13
Stratagene........................................................................ 14
Techne.............................................................................. 14
Bio-Rad Laboratories......................................................... 15
Low-Throughput qPCR Systems............................................. 16
Cepheid............................................................................ 16
Corbett Research............................................................... 16
Roche Applied Science...................................................... 16
qPCR Applications Guide....................................................... 17
Quality of the Template......................................................... 17
Level of Quantitative PCR Controls........................................ 17
Absolute Quantitation....................................................... 17
Relative Quantitation......................................................... 17
Qualitative Analysis........................................................... 17
Summary.......................................................................... 17

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Introduction
The routine study of DNA became practical with the Numerous qPCR detection chemistries and instruments
invention of the polymerase chain reaction (PCR) by are now available to answer a wide range of questions.
Kary Mullis in 1983. With the advent of PCR, it was For instance, qPCR can be used to measure viral load or
possible to multiply a given DNA segment from com- bacterial pathogens in a clinical sample, to verify

qPCR Technical Guide

plex genetic material millions of times in a few hours microarray data, for allelic discrimination or to deter-
using simple equipment. PCR provided researchers with mine RNA (via cDNA) copy numbers to analyze the level
the ability to generate enough genetic material to study of gene product in a tissue sample.
gene function and the effects of mutations, offering The Quantitative PCR Technical Guide from Sigma-Aldrich
new possibilities in basic research and diagnostics. is intended to provide new users with an introduction
Despite these advances, quantitation of DNA or RNA in to qPCR, an understanding of available chemistries, and
cells remained a difficult task until 1993 when Russell the ability to apply qPCR to answer research questions.
Higuchi, et al.,1 introduced real-time, or kinetic, moni- The guide also contains numerous tips and tools for the
toring of DNA amplification. Higuchi’s experiments experienced qPCR user.
revealed that the relationship between the amount of
target DNA and the amount of PCR product generated
after a specific number of amplification cycles is linear.
This observation formed the basis for real-time quanti-
tative PCR (qPCR).

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 Quantitative PCR: How does it work?
Real Time quantitative PCR (qPCR) is very similar to traditional There are two ways to graph qPCR fluorescence data: a standard
PCR. The major difference being that with qPCR the amount of X-Y plot of fluorescence versus cycle number and a semi-log plot
PCR product is measured after each round of amplification while of log fluorescence versus cyle number (Fig. 1). Since PCR is a
with traditional PCR, the amount of PCR product is measured geometric amplification, ideally doubling every cycle, a linear plot
Quantitative PCR: How does it work?

only at the end point of amplification. of the data should show a classic exponential amplification as it
does in the standard X-Y plot. A logarithmic plot of a successful
The concept of qPCR is simple: amplification products are measured
geometric reaction will result in a straight line in the exponential
as they are produced using a fluorescent label. During amplification,
region of the graph. The slope of this portion of the semi-log plot
a fluorescent dye binds, either directly or indirectly via a labeled
can be used to calculate the efficiency of the PCR.
hybridizing probe, to the accumulating DNA molecules, and
fluorescence values are recorded during each cycle of the amplifi- Both plots can be broken into different regions showing the phases
cation process. The fluorescence signal is directly proportional to of PCR amplification. The different graphing techniques empha-
DNA concentration over a broad range, and the linear correlation size different reaction phases. During a typical qPCR experiment,
between PCR product and fluorescence intensity is used to calculate the initial concentration of template is extremely low; therefore
the amount of template present at the beginning of the reaction. the resulting product-related fluorescence is too low to be detected.
The point at which fluorescence is first detected as statistically The background signal is shown as baseline in Figure 1. After the
significant above the baseline or background, is called the threshold yield has reached the detection threshold, shown as the dotted
cycle or Ct Value. line, the reaction course can be followed reliably through the
exponential phase, which is best tracked in the semi-log plot.
The Ct Value is the most important parameter for quantitative PCR.
Once the reaction reaches significant product inhibition, or limiting
This threshold must be established to quantify the amount of
reagent, the reaction reaches a linear phase, which is best tracked
DNA in the samples. It is inversely correlated to the logarithm of
in the linear plot. After this point, the reaction is at the maximum
the initial copy number. The threshold should be set above the
yield, or the plateau phase.
amplification baseline and within the exponential increase phase
(which looks linear in the log phase). Most instruments automati- There are two main methods used to perform quantitative PCR:
cally calculate the threshold level of fluorescence signal by dye-based, or non-specific detection, and probe-based, or specific
determining the baseline (background) average signal and setting detection. Both methods rely on calculating the initial (zero cycle)
a threshold 10-fold higher than this average. DNA concentration by extrapolating back from a reliable
fluorescent signal.
In theory, an equal number of molecules are present in all of the
reactions at any given fluorescence level. Therefore, at the
threshold level, it is assumed that all reactions contain an equal
number of specific amplicons. The higher the initial amount of
sample DNA, the sooner the accumulated product is detected in
the fluorescence plot, and the lower the Ct value.

Figure 1. PCR Amplification Plots

Standard X-Y Plot Semi-log Plot

Plateau
Plateau 0 Linear
1
Log (Relative Fluorescence)
Relative Fluorescence

0.75 –1
Linear
Exponential
0.5
–2

0.25
–3 Baseline Cycle threshold
Baseline Exponential Ct ≅ 15.5
0
10 20 30 40 10 20 30 40
Cycle Number Cycle Number

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qPCR Detection Methods

Both dye-based and probe-based qPCR detection methods utilize binding dye SYBR® Gold, to DNA. A selection of commonly used
a fluorescent signal to measure the amount of DNA in a sample. DNA binding dyes is presented in Table 1.
Fluorescence results from the molecular absorption of light energy
Table 1. DNA Binding Dyes
by fluorescent compounds, fluorophores, at one wavelength and

qPCR Detection Methods

the nearly instantaneous re-emission at another, longer wavelength DNA Binding Dyes
of lower energy. The fluorescence signature of each individual SYBR® Green I
fluorophore is unique in that it provides the wavelengths and
amount of light absorbed and emitted. During fluorescence, the SYBR® Gold
absorption of light excites electrons to a higher electronic state YoYo™-1
where they remain for about 1-10 × 10–8 seconds and then they Yo-Pro™-1
return to the ground state by emitting a photon of energy. The
BOXTO
intensity of the emitted light is a measure of the number of photons
emitted per second. BEBO
Most fluorophores are either heterolytic or polyaromatic hydro- Amplifluor™
carbons. Fluorescence intensity depends on the efficiency with Quencher-labeled primers I
which fluorophores absorb and emit photons, and their ability to Quencher-labeled primers II
undergo repeated excitation/emission cycles. The maximal absorption
and emission wavelengths, and the excitation coefficients, of the LUX™ primers
most common fluorophores are listed in Appendix 1: Traits of Prior to binding DNA, these dyes exhibit low fluorescence. During
Common Fluorophores. amplification, increasing amounts of dye bind to the double-
Figure 2 below shows the emission curves of a selection of stranded DNA products as they are generated. For SYBR Green I,
common fluorophores. after excitation at 497 nm (SYBR Gold 495 nm), an increase in
emission fluorescence at 520 nm (SYBR Gold 537 nm) results
during the polymerization step followed by a decrease as DNA is
Dye-Based Detection denatured. Fluorescence measurements are taken at the end of
the elongation step of each PCR cycle to allow measurement of
Dye-based detection is performed via incorporation of a DNA
DNA in each cycle. See Figure 3, for an illustration of how a dye
binding dye in the PCR. The dyes are non-specific and bind to any
based assay works. Assays using SYBR Green I binding dye are less
double-stranded DNA (dsDNA) generated during amplification
specific than conventional PCR with gel detection because the
resulting in the emission of enhanced fluorescence. This allows
specificity of the reaction is determined entirely by the primers.
the initial DNA concentration to be determined with reference
However, additional specificity can be achieved and the PCR can
to a standard sample.
be verified by melt or dissociation curves.
DNA Binding Dyes – How They Work Melt/Dissociation Curves
DNA binding dyes bind reversibly, but tightly, to DNA by intercalation,
Melt curves allow a comparison of the melting temperatures of
minor groove binding, or a combination of both. Most real-time
amplification products. Different dsDNA molecules melt at
PCR assays that use DNA binding dyes detect the binding of the
different temperatures, dependent upon a number of factors
fluorescent binding dye SYBR® Green I, or the more stable
including GC content, amplicon length, secondary and tertiary

Figure 2. Fluorescent Emission of Common Fluorophores

Emission Curves
1000
900
800
Fluorescence (au)

700
600
500
400
300
200
100
0
100 390 410 430 450 470 490 510 530 550 570 590 610 630 650 670 690 710

Wavelength (nm)

■ Alexa dye (346) ■ FAM (420) ■ TET ■ JOE (500) ■ Cy3 (520) ■ Cy3 (528) ■ REX (520)
■ Tamra ■ RR (540) ■ ROX dye (550) ■ BTR (550) ■ TR (550) ■ Cy5

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 qPCR Detection Methods
Figure 3. Dye-Based qPCR using SYBR Green I Binding Dye
1) The PCR reaction contains enzyme, 2) As the reaction progresses, double- 3) When enough products have
dNTPs, buffer and SYBR Green I, stranded products are generated. The accumulated the fluorescence rises
primers and template. SYBR Green I SYBR Green I intercalcates into these above background. This is called the
qPCR Detection Methods

has no significant fluorescence in the products and begins to fluoresce. threshold cycle of Ct. The Ct value is
presence of single-stranded DNA. used to quantitate the starting
. amount of template.
Extension and Extension and ExtensionDenaturation
and Denaturation Denaturation
SYBR Green SYBR Green SYBR Green
JumpStart intercalation intercalation and reannealing
intercalation and reannealing and reannealing


JumpStart JumpStart





ReadyMix ReadyMix ReadyMix
 primer
 primer  primer

template template

template  
Annealing Annealing Annealing   


   
 
Extension and Extension and Extension and
intercalation intercalation intercalation


  

  

structure, and the chemical formulation of the reaction chemistry. n Are an affordable and ideal method for optimizing qPCR reactions
To produce melt curves, the final PCR product is exposed to a n Uses conventional PCR primers
temperature gradient from about 50 °C to 95 °C while fluorescence
n Does not require an expensive probe to identify a specific target
readouts are continually collected. This causes denaturation of all
dsDNA. The point at which the dsDNA melts into ssDNA is observed n Are useful for generating melt curves, providing an economical
as a drop in fluorescence as the dye dissociates. The melt curves are solution for genotype analysis
converted to distinct melting peaks by plotting the first negative Disadvantages
derivative of the fluorescence as a function of temperature (-dF/dT). n Bind non-specifically to any double-stranded DNA
Products of different lengths and sequences will melt at different
temperatures and are observed as distinct peaks. It is important to n Cannot be used to compare levels of different targets
note that the populations are not necessarily homogeneous and Some of the DNA binding dyes will bind to single-stranded DNA
may contain multiple PCR product species. However, if the PCR (ssDNA). They also bind indiscriminately to any dsDNA, resulting
assay is fully optimized, it is possible to produce a melting profile in non-specific fluorescence and overestimation of the actual
that contains only a single peak representing the specific product product. Non-specific binding results in fluorescence readings in
expected from the primer pair. In this situation, SYBR Green I may the “no template controls” (NTC) due to dye molecules binding
be useful for mutation detection as amplicons that differ by a single to primer dimers and misprimed products. For RT-PCR assays,
nucleotide will melt at slightly different temperatures and can be separate reverse-transcription, PCR, and DNase treatment can
distinguished by their melting peaks. This makes it possible to dramatically reduce non-specific priming and provide more
distinguish homozygotes, a single peak, from heterozygotes, two accurate quantification when using SYBR Green I binding dye.
peaks.2 Please see www.corbettresearch.com for further
Another concern is that multiple dye molecules may bind to a
information about high resolution melt analysis for genotype
single amplified molecule. Signal intensity is dependent on the
analysis using dsDNA binding dyes.
mass of dsDNA in the reaction. Even in reactions with the same
Advantages and Disadvantages of amplification efficiencies, amplification of a longer product will
Dye-Based Detection generate more signal than a shorter one, leading to the implica-
Advantages tion that there are more copies of the longer template than the
shorter one. If amplification efficiencies are different, quantifica-
n Arethe most economical format for detection and quantifica-
tion of PCR products tion will be even more inaccurate.

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 INNOVATION @ WORK

Probe-Based Detection The DNA polymerase then displaces the Reporter molecule from the probe
resulting in fluorescence. The fluorescence accumulates as cycling of PCR contin-
Probe-based quantitation uses sequence specific DNA-based ues and is measured at the end of each PCR cycle. The intensity of fluorescence
generated by the Reporter molecule above background level (Ct value) is mea-
fluorescent reporter probes. Sequence specific probes result in
sured and used to quantitate the amount of newly generated double-stranded
quantification of the sequence of interest only and not all dsDNA.

qPCR Detection Methods

DNA strands.
The probes contain a fluorescent reporter and a quencher to Replicated DNA: After repeating the denaturation, annealing and extension
prevent fluorescence. Common fluorescence reporters include cycles approximately 35-40 times, analysis can begin. The Ct values can be used
to quantitate starting amounts of DNA or to establish a standard curve for gene
derivatives of fluorescein, rhodamine and cyanine. Quenching is
expression studies or other comparative analysis.
the process of reducing the quantum yield of a given fluorescence
process. Quenching molecules accept energy from the fluorophore Probe-based quantitation can also be used for multiplexing:
and dissipate it by either proximal quenching or by Fluorescence detecting multiple targets in a single reaction. Different probes
Resonance Energy Transfer (FRET, see Fig. 4).3 Most reporter systems can be labeled with different reporter fluorophores allowing the
utilize FRET or similar interactions between the donor and quencher detection of amplification products from several distinct
molecules in order to create differences in fluorescence levels sequences in a single qPCR reaction. See Appendix 2 for an
when target sequences are detected. The fluorescent reporter Applications Note on Multiplexing.
and the quencher are located in close proximity to each other in
order for the quencher to prevent fluorescence. Once the probe Many probe-based detection systems are available and they are
locates and hybridizes to the complementary target, the reporter all variations on a single theme with significant differences in
and quencher are separated. The means by which they are separated complexity, cost, and the results obtained. Therefore, it is
varies depending on the type of probe used. Separation relieves important that the chemistry selected is appropriate for the
quenching and a fluorescent signal is generated. The signal is intended application. For an overview of available specific
then measured to quantitate the amount of DNA. The main chemistries, see Table 2.
advantage to using probes is the specificity and sensitivity they Chapter 3 contains additional information on available probes
afford. Their major disadvantage is cost. and primers for specific quantitation to aid in the understanding
and selection of the ideal chemistry.
Figure 4. Fluorescence Resonance Energy Transfer
Table 2. Probe-Based Chemistries and Modifications
Probe-Based Chemistries
Linear Probes Structured Probes
Hydrolysis probes Molecular Beacons
Hybridization probes Scorpions™
Probe Modifications
Locked Nucleic Acids (LNA) Minor Groove Binders (MGB)

Denaturation: Double-stranded DNA is heated to 94 °C-98 °C. During this period,

the double-stranded DNA helix melts open into two single-stranded templates.
Annealing: The reaction is cooled to 45 °C-65 °C. Probes labeled with both
a Reporter molecule and a Quencher molecule and sequence-specific primers
anneal to the single-stranded DNA template. During this cycle, DNA polymerase
attaches to the primed template and begins to incorporate complementary
nucleotides (dATP, dCTP, dGTP, TTP). This process is very slow because the poly-
merase is inefficient at these lower temperatures.
Extension: The temperature is raised slightly during the extension cycle to
65 °C-75 °C. The optimal temperature for Taq DNA polymerase is 72 °C.
During this phase, DNA polymerase extends the sequence-specific primer with
the incorporation of nucleotides that are complementary to the DNA template.

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 Primer and Probe Design
Various primer and probe formats are available for performing Figure 5. Function of Hydrolysis Probes
qPCR assays. The vast majority of assays use the 5’ nuclease
format. This format functions by release and generation of a
fluorescent signal due to the inherent nuclease activity of the
Primer and Probe Design

polymerase used. The assay requires a pair of PCR primers and a

probe labeled with 5’ reporter and 3’ quencher molecules. While
this method is the most widely used, there are several other
formats available that may offer advantages in certain situations.
Examples include the SYBR Green intercalating dye detection
method, the Molecular Beacon technology and probes with a
more complex structure, such as the Scorpions. Finally, increasing
use is also being made of modifications in primer and probe
design. Locked Nucleic Acids® (LNAs) and Minor Groove Binders
(MGB™) provide significant advantages in certain assays that may
not be as amenable to the more traditional approaches. The
methods available fall into two different categories: linear probes
and structured probes.

Linear Probes Advantage: This is the most popular qPCR chemistry and relies
The major advantages of linear probes are that the absence of on the activity of Taq DNA polymerase.
secondary structure allows for optimum hybridization efficiency, Uses: Undoubtedly the chemistry of choice for most quantifica-
and they are extremely simple to design and use. The most tion applications and for those requiring multiplexing.
common linear probes are described below. All probes included in
this section are FRET based. Hybridization Probes
Developed by: Developed specifically for use with the Idaho
Hydrolysis Probes Technology/Roche capillary-based instrument, but can be used
Also known as Dual Labeled Fluorescent Probes (DLFP) or TaqMan® with many real-time instruments.
Developed by: Roche Molecular Systems How They Work: Two probes are designed to bind adjacent to
How They Work: The TaqMan method relies on the 5’-3’ one another on the amplicon. One has a donor dye at its 3’ end,
exonuclease activity of Thermus aquaticus (Taq) DNA polymerase FAM for example. The other has an acceptor dye on its 5’ end,
to cleave a labeled probe when it is hybridized to a complemen- such as LightCycler® Red 640 or 705, and is blocked at its 3’ end
tary target. A fluorophore is attached to the 5’ end of the probe to prevent extension during the annealing step. Both probes
and a quencher to the 3’ end. If no amplicon complementary to hybridize to the target sequence in a head-to-tail arrangement
the probe is present, the probe remains intact and low fluores- during the annealing step. The reporter is excited and passes its
cence is detected. If the PCR results in a complementary target, energy to the acceptor dye through FRET and the intensity of the
the probe binds to it during each annealing step of the PCR. The light emitted is measured by the second probe. Figure 6 illustrates
double-strand-specific 5’-3’ nuclease activity of the Taq enzyme how these probes work.
displaces the 5’ end of the probe and then degrades it. This process
releases the fluorophore and quencher into solution, spatially Figure 6. Function of Hybridization Probes
separating them, and leads to an irreversible increase in fluores-
bv
cence from the reporter. Reaction conditions must be controlled bv

to ensure that the probe hybridizes to the template prior to

elongation from primers. The probe is usually designed to hybridize Primer Donor Probe Acceptor Probe
5’ 5’ P
at 8-10 °C above the Tm of the primers and to perform the
elongation step at a lower temperature to ensure maximum 5’

5’-3’ exonuclease activity of the polymerase. Since this also

Hybridization probes produce fluorescence when both are annealed to
reduces enzyme processivity, short amplicons are designed. a single strand of amplification product. The transfer of resonance energy
Figure 5 illustrates how these probes work. from the donor fluorophore (3’-fluorescein) to the acceptor fluorophore
(5’-LC Red 640) is a process known as fluorescence resonance energy transfer.

Advantages: Since the probes are not hydrolyzed, fluorescence

is reversible and allows for the generation of melt curves.
Uses: Can be used for SNP/mutation detection, where one probe
is positioned over the polymorphic site and the mismatch causes
the probe to dissociate at a different temperature to the fully
complementary amplicon.4 As stated above, they can also be
used to generate melt curves.

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Structured Probes Uses: Molecular Beacons7 probes have become popular for
standard analyses such as quantification of DNA and RNA.8 They
Thermodynamic analysis reveals that structurally constrained have also been used for monitoring intracellular mRNA hybridiza-
probes have higher hybridization specificity than linear probes.5,6 tion,9 RNA processing,10 and transcription11 in living cells in real-
Structurally constrained probes also have a much greater

Primer and Probe Design

time. They are also ideally suited to SNP/mutation analysis12 as
specificity for mismatch discrimination due to the fact that in the they can readily detect single nucleotide differences13,14 and have
absence of target they form fewer conformations than unstruc- been reported to be reliable for analysis of G/C-rich sequences.15
tured probes, resulting in an increase in entropy and the free The sensitivity of Molecular Beacons probes permits their use for
energy of hybridization. The following outlines the traits of the the accurate detection of mRNA from single cells.16 They have
two most common structured probes. been used in fourplex assays to discriminate between as few as
Molecular Beacons Probes 10 copies of one retrovirus in the presence of 1 × 105 copies of
another retrovirus.17
Developed by: Public Health Research Institute in New York
How They Work: They consist of a hairpin-loop structure that
Scorpions™ Probes
forms the probe region and a stem formed by annealing of Developed by: DsX, Ltd.
complementary termini. One end of the stem has a reporter How They Work: The Scorpions uni-probe consists of a single-
fluorophore attached and the other, a quencher. In solution, the stranded bi-labeled fluorescent probe sequence held in a hairpin-
probes adopt the hairpin structure and the stem keeps the arms in loop conformation (approx. 20 to 25 nt) by complementary stem
close proximity resulting in efficient quenching of the fluorophore. sequences (approx. 4 to 6 nt) on both ends of the probe. The
During the annealing step, the probe is excited by light from the probe contains a 5’-end reporter dye and an internal quencher
PCR instrument (hu1). Molecular Beacons hybridize to their target dye directly linked to the 5’-end of a PCR primer via a blocker.
sequence causing the hairpin-loop structure to open and separate The blocker prevents Taq DNA polymerase from extending the
the 5’-end reporter dye from the 3’-end quencher. The quencher PCR primer. The close proximity of the reporter dye to the
is no longer close enough to absorb the emission from the quencher dye causes the quenching of the reporter’s natural
reporter dye. This results in fluorescence of the dye and the PCR fluorescence.
instrument detects the increase of emitted energy (hu2). The At the beginning of the real-time quantitative PCR reaction, Taq
resulting fluorescent signal is directly proportional to the amount DNA polymerase extends the PCR primer and synthesizes the
of target DNA. If the target DNA sequence does not exactly match complementary strand of the specific target sequence. During the
the Molecular Beacon probe sequence, hybridization and next cycle, the hairpin-loop unfolds and the loop region of the
fluorescence does not occur. When the temperature is raised to probe hybridizes intra-molecularly to the newly synthesized target
allow primer extension, the Molecular Beacons probes dissociate sequence. The reporter is excited by light from the real-time
from the targets and the process is repeated with subsequent quantitative PCR instrument (hu1). Now that the reporter dye is
PCR cycles. See Figure 7 for an illustration of how these probes no longer in close proximity to the quencher dye, fluorescence
work. emission may take place (hu2). The significant increase of the
fluorescent signal is detected by the real-time PCR instrument
Figure 7. Function of Molecular Beacons Probes and is directly proportional to the amount of target DNA. See
Figure 8 for an illustration of how these probes work.

Figure 8. Function of Scorpions Probes

Advantage: Greater specificity for mismatch discrimination due

to structural constraints.
Disadvantage: The main disadvantage associated with Molecular
Beacons is the accurate design of the hybridization probe. Optimal
design of the Molecular Beacon stem annealing strength is crucial. Scorpions bi-probe, or duplex Scorpions probes, were developed
This process is simplified with the use of specific software packages to increase the separation of the fluorophore and the quencher.
such as Beacon Designer from Premier Biosoft or by contacting The Scorpions bi-probe is a duplex of two complementary labeled
a member of the design team at www.designmyprobe.com oligonucleotides, where the specific primer, PCR blocker region,
for assistance. probe, and fluorophore make up one oligonucleotide, and the
quencher is linked to the 3’-end of a second oligonucleotide that

Our Innovation, Your Research — Shaping the Future of Life Science 7

 Primer and Probe Design
is complementary to the probe sequence. The mechanism of in 3’-endo conformation restricting the flexibility of the ribofura-
action is essentially the same as the uni-probe format.18 This nose ring and locking the structure into a rigid bicyclic formation.
arrangement retains the intramolecular probing mechanism This structure confers enhanced hybridization performance and
resulting in improved signal intensity over the standard Scorpions exceptional biological stability.
Primer and Probe Design

uni-probe format. See Figure 9 for an illustration of how

Scorpions bi-probes work. LNA Monomer
Base
Figure 9. Function of Scorpions Bi-Probes HO
HO
O
O
O O Base

Advantages
n Increased thermal duplex stability22
n Improved specificity of probe hybridization to target sequence23
n Enhanced allelic discrimination
n Added flexibility in probe design
Advantages: Scorpions probes combine the primer and probe in n Compatible with many systems
one molecule converting priming and probing into a unimolecular Increased Stability and Improved Specificity
event. A unimolecular event is kinetically favorable and highly
The LNA monomer chemical structure enhances the stability of
efficient due to covalent attachment of the probe to the target
the hybridization of the probe to its target. As a result, the duplex
amplicon ensuring that each probe has a target in the vicinity.19
melting temperature, Tm, may increase by up to 8 °C per LNA
Enzymatic cleavage is not required so the reaction is very rapid.
monomer substitution in medium salt conditions compared to a
This allows introduction of more rapid cycling conditions combined
DNA fluorescent probe for the same target sequence, depending
with a significantly stronger signal compared to both TaqMan
on the target nucleic acid.24 This increase in hybridization creates
probes and Molecular Beacons probes.20 Another advantage over
a significant broadening in the scope of assay conditions and
TaqMan assays is that the PCR reaction is carried out at the
allows for more successful single-tube multiplexing.25 Addition-
optimal temperature for the polymerase rather than at the
ally, this benefit also makes it possible to optimize the Tm level
reduced temperature required for the 5’-nuclease activity to
and thus the hybridization specificity via placement of the LNA
displace and cleave the probe. The most important benefit is that
base(s) in the probe design.26 By increasing the stability and the
there is a one-to-one relationship between the number of
specificity, background fluorescence from spurious binding is
amplicons generated and the amount of fluorescence produced.
reduced and the signal-to-noise ratio is increased.
Uses: Scorpions probes are ideally suited to SNP/mutation
detection and have been used to detect, type and quantitate Enhanced Allelic Discrimination
human papillomaviruses.21 SNP detection can be carried out LNAs can also be used for allelic discrimination. They provide an
either by allele specific hybridization or by allele specific exten- extremely reliable and effective means for SNP-calling in genotyp-
sion. If the probe sequence is allele-specific, allelic variants of a ing applications. The presence of a single base mismatch has a
SNP can be detected in a single reaction by labeling the two greater destabilizing effect on the duplex formation between a
versions of the probe with different fluorophores. Alternatively, LNA fluorescent probe and its target nucleic acid than with a
the PCR primer can be designed to selectively amplify only one conventional DNA fluorescent probe. Shorter probes incorporat-
allele of a SNP. Results with Scorpions probes compare favorably ing LNA bases can be used at the same temperatures as longer
with the high signal/high background ratio of the TaqMan probes conventional DNA probes.
and low signal/low background ratio of Molecular Beacons probes. Added Flexibility in Probe Design
Due to the enhanced hybridization characteristics and the Tm
Modifications contribution, LNA containing qPCR probes can be synthesized to
be shorter, allowing flexibility in design while still satisfying assay
A number of modifications can be incorporated when designing design guidelines. As such, certain design limitations that cannot
probes to provide enhanced performance. Locked Nucleic Acids be overcome with standard DNA chemistries can be reduced or
and Minor Grove Binders are two of the most commonly used eliminated. For instance, shorter probes can be designed to
modifications. address traditionally problematic target sequences, such as AT- or
GC-rich regions. LNAs also facilitate the querying of difficult or
inaccessible SNPs, such as the relatively stable G:T mismatch.
Locked Nucleic Acid
A Locked Nucleic Acid (LNA) is a novel type of nucleic acid analog
that contains a 2’-O, 4’-C methylene bridge. The bridge is locked

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 INNOVATION @ WORK

LNAs can also be used when AT-rich qPCR probes need to be over
30 bases long to satisfy amplicon design guidelines. With LNA
Template, Primer Design,
fluorescent probes, the selective placement of LNA base substitu- Probe Design and Dye Choice
tions facilitates optimal design of highly specific, shorter probes
It is very important to ensure that the primers and probes are

Primer and Probe Design

that perform very well, even at lengths of 13-20 bases.
optimally designed in order to perform successful quantitative
Compatible with Many Platforms PCR. There are many points in the design process that must be
LNA probes are also compatible with real-time PCR platforms and considered: the template, primer design, probe design, dye
end-point analytical detection instruments depending on the choice in probe design, quenchers in probe design, and the
excitation/emission wavelengths of the dyes and of the equip- software the system uses.
ment. This provides the ability to work with the instrumentation Template Considerations
and reagents of choice under universal cycling conditions.
1. Amplicons should ideally be between 50-150 bases
in length.
Minor Groove Binders Shorter amplicons tend to be more tolerant of less than ideal
reaction conditions and allow probes to successfully compete
Minor Groove Binders (MGBs) are a group of naturally occurring with the complementary strand of the amplicon. This allows
antibiotics. They are long, flat molecules that can adopt a crescent for faster and more efficient reactions and increased consis-
shape allowing them to bind to duplex DNA in the minor groove. tency of results.
MGBs are stabilized in the minor groove by either hydrogen bonds
2. For RNA targets, select primers spanning exon-exon
or hydrophobic interactions. This results in the stabilization of the
junctions.
probe-DNA hybridization.
By choosing primers that span exon-exon junctions, amplifica-
MGBs produce a “Tm leveling” effect as A/T content increases. tion of contaminating genomic DNA in cDNA targets can
Natural 8-mer probes exhibit a linear decrease in Tm as A/T be avoided.
content increases while MGB probes exhibit level Tm as A/T
3. Consideration of template secondary structure is
content increases. The increase in melting temperature allows for
important.
the use of shorter probes with improved mismatch discrimination.
Close attention should be paid to template secondary structure.
Advantages This is one area where qPCR primer and probe design differs
n Useful for functional assays for difficult targets slightly from traditional PCR design. In traditional PCR design,
n Higher accuracy and confidence in results template secondary structure is not necessarily a critical factor.
Templates containing homopolymeric stretches of greater than
n Compatible with any real-time PCR detection system four consecutive bases should be avoided. Significant second-
Functional Assays for Difficult Targets ary structure may hinder primers from annealing and prevent
complete product extension by the polymerase. This is a
There are many sequence features and applications that pose
particular concern in cases of stretches of Gs. A useful tool for
problems when trying to design probes and primers for specific
the assessment of template secondary structure in DNA targets
targets. For example, sequential “Gs” result in secondary
is the Mfold server, developed by Dr. Michael Zuker and
structure that can block efficient hybridization, weaker bonds
maintained at the Rensselaer and Wadsworth Bioinformatics
within “A/T” rich regions result in lower melting temperatures
Center. (http://www.bioinfo.rpi.edu/applications/mfold/)
(Tm) that inhibit the use of efficient PCR conditions, and limited
target regions can restrict the length of optimal probe and primer Primer Design Considerations
sequences impacting their functionality. Since MGBs allow for 1. Avoid secondary structure problems.
design of shorter probes and for the design of specific Tms, they When feasible, one should attempt to pick regions of the
aid in the success of assays posing these difficulties. template that are not apt to cause secondary structure
Higher Accuracy and Confidence in Results problems. For more information, please refer to number 3
under “Template Design Considerations.”
Since shorter probes can be used, mismatch sensitivity can be
increased with the use of MGBs. Also, since MGB probes have 2. Avoid runs of Gs and Cs longer than three bases.
more stable hybridization characteristics and remain intact after In the primer sequences, runs of Gs and Cs longer than three
PCR, they are not cleaved by the 5’-nuclease activity of Taq DNA bases should be avoided. This is very important at the 3’-end
polymerase. As a result, more symmetric melting curves using the of a primer where such runs may result in a phenomenon
same probe and primer set in the same tube as the real-timer PCR referred to as polymerase slippage, which can be a problem in
experiments can be produced to confirm PCR and detection specificity. standard PCR. Polymerase slippage occurs during replication
when Taq polymerase slips from the DNA template strand at
Compatible with Any Real-Time PCR Instrument
the repeat region and then reattaches at a distant site. This can
MGB Eclipse probe systems will work on any manufacturer’s real- cause a new DNA strand to contain an expanded section of DNA.
time or end-point analytical instrumentation. The probes have
been tested on most leading systems.

Our Innovation, Your Research — Shaping the Future of Life Science 9

 Primer and Probe Design
3. Aim to design primers with a Tm higher than the Tm of modifying bases, or Locked Nucleic Acid® (LNA®) fluorescent
any of the predicted template secondary structures. probes may be beneficial. LNA fluorescent probes are modified
To be sure that the majority of possible secondary structures RNA nucleotides that exhibit thermal stabilities towards
have been unfolded before the primer-annealing step, one complementary DNA and RNA. LNA probes serve to increase
Primer and Probe Design

should also aim to design primers with a melting temperature, the thermal stability and hybridization stability, allowing for
Tm, higher than the Tm of any of the predicted template more accurate gene quantitation and allelic discrimination and
secondary structures. Again, the Mfold server is a very useful providing easier and more flexible probe designs for problem-
resource in this phase of the design process. atic target sequences.
4. Design primers longer than 17 bases. 3. Probe GC content may be anywhere from 30-80%
Shorter primers increase the chance of random, or non- preferably with a greater number of Cs than Gs.
specific, primer binding. Such random primer binding may be As in the case of primers, there should not be homopolymeric
more marked in targets of higher complexity, such as genomic runs of single bases, most definitely not of Gs, in probes.
DNA, and should be guarded against even when using more
4. For optimal efficiency of dual-labeled probes, the
predictable target sequences, such as plasmids. Even with a
5’-terminal of the probe (carrying the reporter dye)
plasmid, it is not always the case that the sequence is
should be as close as possible to the 3’-end of the
completely known.
forward primer.
5. Check the overall specificity of primers (and probes) by For Molecular Beacons probes, the probe should be designed
carrying out a BLAST search. to anneal in the middle of the amplicon.
BLAST, or Basic Local Alignment Search Tool, searches can be
5. A G placed at the extreme 5’ end of the probe adjacent
performed using the resources of the publicly available site at
to the reporter dye should be avoided.
NCBI at http://www.ncbi.nlm.nih.gov/. A BLAST search
This may lead to spontaneous fluorophore quenching.
compares primer sequences against a library of genomic DNA
sequences. Another method is to use electronic PCR, or e-PCR. Dye Choice in Probe Design
e-PCR is used to identify sequence tagged sites (STS) within DNA It is also important to consider the dye choice when designing
sequences by searching for sub-sequences that closely match probes.
the PCR primers. STSs are short DNA segments occurring once
1. Make sure that the instrument being used can detect
in the human genome. Their exact location and sequence are
the dyes.
known and, as a result, they can serve as landmarks in the
human genome. e-PCR uses UniSTS, a database of STSs, to 2. When designing fluorescent probes, it is important to be
identify primers that have the proper order that could represent sure that the fluorophore and quencher are compatible.
PCR primers used to generate the known STSs. This tool is 3. When designing multiplexed reactions, spectral overlap
available at http://www.ncbi.nlm.nih.gov/sutils/e-pcr/. should be minimized.
6. Forward and reverse PCR primers should be analyzed for Table 3 provides the excitation wavelength and the emission
self-complementarity in their sequences. wavelength for several popular dyes used in probe design.
In particular, 3’ self-complementarity primer-dimer formation
should be avoided. A number of commercial primer analysis Table 3. Dye Choice in Probe Design
resources are available to aid in this process. Filter Excitation Wv Emission Wv
7. Multiplexed primer pairs must all work efficiently at the Alexa 350 350 440
same annealing temperature.
FAM/SYBR Green 492 516
Probe Design Considerations TET 517 538
1. Probes may be anywhere from 9-40 bases in length.
HEX/JOE/VIC 535 555
In the case of the 5’-nuclease assay using dual-labeled probes,
probes with a reporter and a quencher, the overall probe Cy3 545 568
length tends to range up to 30 bases. Longer probes might TAMARA 556 580
compromise the efficiency of signal quenching. In the case of
ROX/Texas Red 585 610
Molecular Beacons probes, where quenching is a function of
the self-annealing hairpin design, probes may be a little longer, Cy5 635 665
perhaps up to 35 bases.
2. In most situations, the probe Tm should be approxi- Quenchers in Probe Design
mately 10 ºC higher than the Tm of the primers. FRET occurs when donor and acceptor molecules are separated
This allows for efficient probe-to-target annealing during the by about 100 Å. Since a helix occupies approximately 3.4 Å, the
reaction. During the reaction, the probe should anneal before maximum distance between a reporter and its quencher on a
the primers. When probes anneal before the primers, shorter linear probe should not exceed approximately 30 bases. The
probes can be used. The use of shorter probes is important acceptor can be another fluorophore, in which case the transfer
during mismatch detection. In such cases, the use of special releases the energy from the quencher as fluorescence at a longer

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 INNOVATION @ WORK

wavelength. For instance, the combination of FAM (a fluorescein Primer and Probe Design FAQs
derivative) and TAMRA (a rhodamine derivative) will absorb at What type of probe should I choose?
492 nm (excitation peak for FAM) and emit at 580 nm (emission It depends on the experiment being performed and the machine
peak for TAMRA). The inherent fluorescence and broad emission being used.

Primer and Probe Design

spectrum of TAMRA result in a poor signal-to-noise ratio when this
fluorophore is used as a quencher. This can make multiplexing Table 5 is intended to aid in the selection of probes based on the
difficult. To address this issue, dark quenchers were introduced. desired application.

Dark quenchers absorb the energy emitted by the reporter Table 5. Probe Applications
fluorophore and emit heat rather than fluorescence. Early dark TaqMan/ Molecular
quenchers, such as 4-(49-dimethylaminophenylazo) benzoic acid Application Hydrolysis Hybridization Beacons Scorpions
(DABCYL), had limited spectral overlap between the fluorescent
dye and quencher molecule. Black Hole Quenchers (BHQ™-1 and Quantification X X
BHQ™-2) from Biosearch Technologies have lower background applications
fluorescence and a broad effective range of absorption. As a Multiplexing X X
result, Black Hole Quenchers provide greater sensitivity and SNP/mutation X X X
enable the simultaneous use of a wide range of reporter detection
fluorophores thus expanding the options available for multiplex
Generation of X
assays. It is important to ensure that the Black Hole Quencher is
melt curves
matched with the probe based on the excitation and emission
spectra of the probe. Monitoring X
intracellular
Table 4. Quencher Ranges mRNA
Quencher Quenching Range hybridization,
RNA processing,
BHQ-1 480-580 nm
and transcription
BHQ-2 550-650 nm in living cells
BHQ-3 620-730 nm Analysis of G/C- X
TAMARA 550-576 nm rich sequences
DABCYL 453 nm Detection of X
mRNA from
single cells
Primer and Probe Design Software and Web Sites
Several software programs can be used to design primers and Why do I need to have a good design?
probes, but software is only a tool to aid in the design process Well-designed primers and probes will provide ideal RT-qPCR
and cannot guarantee a perfect design. data: high PCR efficiency, specific PCR products and the most
sensitive results.
Designing Primers
What should I do if no primers and/or probes are found for
n Primer 3.0
my sequence?
Designing TaqMan Probes or Molecular Beacons Probes There are several parameters that can be adjusted to force the
n Beacon Designer from Premier Biosoft program to pick primers and/or probes without significantly
sacrificing primer and/or probe quality.
Designing Scorpions Probes
I have a very short target sequence. Is it possible to design
n DNA Software at dnasoftware.com
an optimal probe?
Designing FRET Probes (LightCycler Probes) It depends on the sequence. To design the probe, it may be
n LightCycler Probe Design Software 2.0 necessary to submit a longer target sequence (up to 160 bases).
Additionally, LNA bases can be included in the probe sequence to
Designing LNA Probes or Oligos reduce the probe length while retaining the optimal characteristic
n A combination of different types of software must be used of the probe.

Our Innovation, Your Research — Shaping the Future of Life Science 11

 Instrumentation
Considerations Dynamic Range
The dynamic range of detection is the difference between the
When Higuchi et. al 1 designed a qPCR system, a conventional
minimum and maximum target concentrations that can be detected.
PCR block, a UV light source and a camera for signal detection
The minimum detectable signal is determined by chemistry and
were the instruments used. Since the design of the first qPCR
Instrumentation

signal-to-noise discrimination of the system. The maximum signal

instrument, there have been many advances in qPCR instrumen-
is restricted by chemistry and the range of signal detection is
tation, but the requirements for a system have remained funda-
determined by the photo detector as well as by the software
mentally the same. When choosing an instrument for qPCR,
analyses capabilities.
it is important to consider the following traits of the system:
Components; Sensitivity; Specificity of Detection; Dynamic Range; Detection Linearity
Detection Linearity; Software. Detection linearity is the concentration range that an instrument
Components is able to linearly measure.
During instrument development, it became clear that certain Software
components of the system required specific traits in order to be For many scientists, the choice of the system will be largely
used for quantitative studies. dependant upon the capabilities of the integrated software.
Conventional PCR thermal blocks were notorious for a lack of Some packages are targeted towards simplicity of analysis with
uniformity that resulted in the “edge effect.” Lack of uniformity little, if any, input from the end user. In contrast, other packages
caused differences in PCR efficiency and product quantity. Since are fully flexible. In the case of full flexibility, every point of raw
this situation is contrary to the needs of quantitative PCR, many data is accessible and the scientist chooses among the extensive
manufacturers invested in unique thermal regulation mechanisms analysis choices. A second consideration is whether having raw
to reduce this effect. fluorescence data would be useful, as in the case of using a
fluorescent thermocycler as a thermostated fluorometer. An
Alternative excitation sources were also developed, with the first
additional consideration is the presentation of data. The manner
source being the laser. The major advantage, and also the disadvan-
in which data is presented varies considerably among the current
tage, of using lasers is that they emit at a single wavelength. A
market offerings. Some manufacturers depend on commercial
single wavelength allows for the specificity of excitation for a
graphics packages while others simply list Ct values.
single dye but does not allow the flexibility to include additional
dyes for multiplexing. The cost and application disadvantages were
resolved by using broad wavelength excitation sources, tungsten Comparing qPCR Instrumentation
bulbs or light emitting diodes (LEDs), in conjunction with specific
light filters. As stated above, it is important to compare systems using exactly
the same target and detection chemistry. When comparing
Many companies have continued to use silicon-based, charge- systems, it is important to perform the following tests:
coupled device (CCD) cameras while others use photo multiplier
tubes (PMTs) as photo detectors. 1. Uniformity–Thermal
a. Run a calibrated end-point PCR and detect on a gel.
While the merits of one hardware design over another can be 2. Uniformity–Optical
debated, the critical factors to consider when selecting a qPCR a. Measure the fluorescence of identical concentrations of dye
system are sensitivity, specificity of detection, dynamic range and aliquoted to every well.
detection linearity. b. Compare the starting fluorescence levels for identical qPCR
Sensitivity probes allocated to every well of the plate.
3. Uniformity–Thermal and Optical
Sensitivity refers to the minimum quantity of target that can be
a. Run identical qPCR experiments in every well and compare
detected above the background noise of the system. Sensitivity is
Ct values.
not only instrument dependent, but is also chemistry dependent.
4. Dynamic Range–Optical
As a result, it is important to carry out comparisons between
a. Plot fluorescence measurements of a serial dye dilution. It
systems using exactly the same target and detection chemistry.
is important to note that while optical dynamic range is
Specificity of Detection significant, qPCR is based upon Ct measurement, which is
Specificity of detection is important at the single dye level because approximated by the minimum above-background fluores-
it is essential that the signal detected is generated specifically cence. This specification is relevant to instruments that can
from the detection chemistry and not from alternative sources or also be used as fluorescent microplate readers.
neighboring reactions on the plate. One significant example is 5. Dynamic Range–Reaction
termed “cross talk.” Cross talk occurs in multiplex reactions when a. Perform qPCR on a serially diluted template (1 copy to
signal from one fluorescent dye is detected as signal from the 1012 copies).
other fluorescent dye in the reaction. 6. Wavelength Discrimination
a. This is the ability to accurately measure fluorescence
intensity from a fluorophore in a mixture of fluorophores.

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On the following pages, qPCR instruments are categorized into more will be considered high throughput. All qPCR instruments
two groups: high throughput and low throughput. Any device require a computer for operation and data management/analysis.
that processes fewer than 96 samples in a run will be called Nearly all instruments are supplied with a computer and the
low throughput and anything that processes 96 samples or appropriate software.

Instrumentation
High-Throughput qPCR Instruments
Applied Biosystems Applied Biosystems Applied Biosystems Roche Applied Science
Manufacturer ABI 7300 Real-Time ABI 7500 Real-Time ABI 7900HT Fast LightCycler® 480
System PCR System PCR System Real-Time PCR System Real-Time PCR System
Advantages Affordable, 5 color multiplexing. Rapid 96- and 384-well plate Interchangeable 96- and
4 color multiplexing cycling upgrade available compatible. The system 384-well thermal blocks.
in the ABI 7500 Fast that can handle TaqMan Low The Therma-Base design
allows run times of <40 Density Arrays with fully provides added tempera-
min. TaqMan assays are automated robotic ture uniformity
available loading. Optional Fast
real-time PCR capability.
TaqMan assays are
available
Excitation Tungsten-halogen lamp, Tungsten-halogen lamp, Extended-life 488 nm Filtered (450, 483, 523,
single-excitation filter 5 excitation filters argon-ion laser is 558 and 615 nm) high-
distributed to all wells by intensity Xenon lamp
a dual-axis synchronous (430–630 nm)
scanning head
Detection 4 emission filters and a 5 emission filters and a CCD camera and a Filters (500, 533, 568,
CCD camera CCD camera spectrograph 610, 640, 670 nm) and a
CCD camera
Heat block Peltier Peltier Peltier Peltier plus Therma-Base
Rxn volume(s): 20-100 µL 25-100 µL 25-100 μL/10-30 μL (fast) 5-100 μL (96 well) or
for 96-well and 5-20 μL 5-20 μL (384 well)
for 384-wells
Multiplexing Up to 4 fluorophores Up to 5 fluorophores Multiplex number is The number is dependent
limited by spectral overlap on the excitation/emission
combination
Sensitivity 10 starting copies of the 10 starting copies of the 10 starting copies of the 10 copies (plasmid)
RNase P gene from human RNase P gene from human RNase P gene from human
genomic DNA genomic DNA genomic DNA
Dynamic Range– 9 9 9 10
Reaction –
Orders of
Magnitude
Software System Software and System Software and System Software and LightCycler Software
Primer Express software Primer Express software Primer Express software
included included included. Enterprise
Edition Software with SNP
Manager and RQ Manager
are available

Our Innovation, Your Research — Shaping the Future of Life Science 13

 Instrumentation
High-Throughput qPCR Instruments
Manufacturer Stratagene Mx3000P® Multiplex Stratagene Mx3005P® Multiplex
System qPCR System qPCR System Techne Quantica®
Instrumentation

Advantages A reference dye is unnecessary A reference dye is unnecessary Up to 4 instruments can be

allowing the use of ROX or Texas allowing the use of ROX or Texas controlled from a single PC. The
Red probes. System can be used Red probes. System can be used system has a robot-friendly CD-type
as a thermostated fluorescent as a thermostated fluorescent block loading mechanism
microplate reader (e.g., to quantify microplate reader (e.g., to quantify
fluorescently stained/labeled nucleic fluorescently stained/labeled nucleic
acids). An embedded computer acids). An embedded computer
safeguards data. The system can safeguards data. The system can
also be linked to a PC allowing also be linked to a PC allowing
multiple systems to be used at the multiple systems to be used at the
same time for high-throughput same time for high-throughput
applications from a single PC applications from a single PC
Excitation Quartz tungsten-halogen lamp Quartz tungsten-halogen lamp Filtered solid-state white light
(470-650 nm)
Detection Scanning fiber optics with four- Scanning fiber optics with four- Photomultiplier tube (500-710 nm)
position filter wheel and a position filter wheel and a
photomultiplier tube photomultiplier tube
Heat block Peltier Peltier Gradient block option with a
gradient range of up to 30 °C
(between 20-70 °C)
Rxn volume(s): 10-60 µL 10-60 µL 15-50 µL
Multiplexing Up to 4 fluorophores Up to 5 fluorophores Up to 4 colors
Sensitivity To a single-copy equivalent To a single-copy equivalent Single copy of template and down
to 1 nM fluorescein
Dynamic Range– 10 10 9
Reaction – Orders
of Magnitude
Software MxPro™ included MxPro™ included. Also includes Quansoft software included
Beacon Designer 4 software

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High-Throughput qPCR Instruments

Bio-Rad Laboratories Bio-Rad Laboratories
Bio-Rad Laboratories MyiQ™ Single-Color DNA Engine Opticon 2® Bio-Rad Laboratories
Manufacturer iQ5 Real-Time PCR Real-Time PCR Real-Time PCR Chromo4™ Real-Time

Instrumentation
System Detection System Detection System Detection System Detector
Advantages Optical upgrade to the Optical upgrade to the Can perform plate reads Has a 96-well Alpha unit.
iCycler® thermal cycler. iCycler thermal cycler. This allows it to be used
Embedded tool for end- Limited to one color on any DNA Engine®
point fluorescence analysis chassis
Excitation Tungsten-halogen lamp Tungsten-halogen lamp 96 LEDs (470-505 nm) 4 LEDs in photonics
(475-645 nm) (475-645 nm) shuttle (450-650 nm)
Detection 5 color customizable 12-bit CCD camera Two photomultiplier 4 photodiodes
filter wheel and CCD (515-545 nm) tubes; one compatible (515-730 nm)
camera (515-700 nm) with SYBR Green I or
FAM; the other detects a
variety of fluorophores
Heat block Peltier and Joule Peltier and Joule Peltier Peltier
(gradient: 25 °C max) (gradient: 25 ºC max) (gradient: 24 ºC max) (gradient: 24 ºC max)
Rxn volume(s): 15-100 µL 15-100 µL 10-100 µL 10-100 µL
Multiplexing Up to 5 colors No Up to 2 colors Up to 4 colors
Sensitivity Linear to 10 copies of One copy of IL-1b in As little as one starting As little as one starting
b-actin DNA human genomic DNA template copy template copy
Dynamic Range– 9 8 10 10
Reaction – Orders
of Magnitude
Software iQ5 Optical System iCycler iQ Software DNA Engine Opticon 2 Chromo4 Software
Software Version 2.0 Version 3.1 Software Version 3.1 Version 3.1

Our Innovation, Your Research — Shaping the Future of Life Science 15

 Instrumentation
Low-Throughput qPCR Systems
Manufacturer Cepheid Corbett Research Corbett Research Roche Applied Science
System SmartCycler® TD System Rotor-Gene™ 3000/3000A Rotor-Gene™ 6000 LightCycler® 2.0
Instrumentation

Advantages Runs samples in blocks Centrifugal systems A centrifugal system The LightCycler series of
of 16, up to 96 samples designed to work with designed to work with instruments are rotor-
total, and allows each any real-time chemistry. any real-time chemistry. based and work with a
reaction to be separately Samples are rotated in Samples are rotated in variety of chemistries.
programmed for inde­ a changing thermal a changing thermal Rapid reactions take
pendent experimental environment to produce environment to produce place in passivated
protocols. Fast programs a uniform temperature a uniform temperature glass capillaries
complete in as little as between reactions. between reactions.
20 minutes. Fast runs Conventional or rapid Conventional or rapid
require proprietary cycling conditions possible cycling conditions possible
reaction tubes using conventional using conventional
reagents reagents. Dedicated
analysis software
Excitation LED 4 LEDs excite entire Separate color high- Blue LED (470 nm)
visible spectrum intensity LED per channel
Detection Silicon photo detector is A series of filters and a Separate emission filter Photodiodes (530, 560,
used to collect filtered photomultiplier tube per channel and a 610, 640, 670 and
light photomultiplier tube 710 nm)
Heat block Solid-state heater; Thermostated air Thermostated air Thermostated air
forced-air cooling
Rxn volume(s): 25 and 100 µL volumes 20-25 µl 5 µL to 100 µL, but 20 µL 20 µL and 100 µL
is typical
Multiplexing Up to 4 colors Two-channel (3000A) or 2, 5, or 6 color options Provides multiplexing and
four channel (3000) measures fluorescence
between 530-705 nm
Sensitivity 10 copies plasmid Unpublished Single-copy gene target Single-copy gene in 3 pg
amplification from a of human genomic DNA.
whole human genome 1-10 copies of plasmid
Dynamic Range– 8 Unpublished Unpublished 10
Reaction – Orders
of Magnitude
Maximum Number of samples can Up to 36 or 72 samples 72 individual tubes or 32
number of be increased in increments in specifically designed heat-sealed plates
samples of 16 – to a total number rotors
of 96 by linking additional
units to a single PC
Software SmartCycler Corbett system software Corbett system software LightCycler® Software 4.05
with analysis, graphing
and statistical license

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qPCR Applications Guide

The popularity of quantitative PCR lies in the many applications Relative Quantitation
for which it can be employed. Common applications include the Relative quantitation requires calculation of the ratio between the
detection of single nucleotide polymorphisms (SNPs), measure- amount of target template and a reference template in a sample.
ment of DNA copy number, determination of genomic allelic The advantage of this technique is that using an internal standard

qPCR Applications Guide

variation, diagnosis and quantitation of bacterial and viral can minimize potential variations in sample preparation and
infections and the validation of gene expression data from handling. The accuracy of relative quantitation depends on the
microarray experiments. In order to perform successful experi- appropriate choice of a reference template for standards.
ments using qPCR, the quality of the template and the level of Variability of the standard will influence the results and so it is
the control must be considered. important that the appropriate standard is used.27 Some
Quality of the Template researchers choose not to run a standard curve and to report
target quantities as a fraction of the reference, a technique
Quantitative PCR can only be used reliably when the template of
termed comparative quantitation. Alternatively, one may assume
interest can be amplified and when the amplification method is
that the amplification efficiencies of the target and reference are
without error. As a result, quality of the template is critical to
negligible and quantify the target based solely on the standard
success. PCR will only amplify DNA or cDNA with an intact
curve determined for the reference sequence. Finally, in the most
phosphodiester backbone between the priming sites. In addition,
accurate of the relative quantitation techniques, the amplification
DNA containing lesions that affect the efficiency of amplification,
efficiencies of both the reference and target are measured and a
such as abasic sites and thymine dimers, will be either under-
correction factor is determined. This process, termed normaliza-
represented or may not be represented at all in qPCR. In order
tion,27 requires examining a sample containing known concentra-
to achieve the best possible quantitation, it is of paramount
tions of both target and reference and the generation of two
importance that the highest quality nucleic acid template goes
standard curves. Since this method measures the amount of
into the qPCR. One factor that may affect the quality of the
target relative to a presumably invariant control, relative qRT-PCR
template is the inclusion of additives that may degrade dNTPs,
is most often used to measure genetic polymorphism differences
primers, and PCR substrates. Some additives can also directly
between tissues or between healthy and diseased samples. It is
inhibit the DNA polymerase thus affecting the results of the
important to note that if using SYBR binding dye-based systems,
qPCR. In addition to using quality template, it is also important
the target and internal reference quantitation must be performed
to include a set of rigorous controls with every experiment to
in separate reactions.
ensure success.
Levels of Quantitative PCR Controls Qualitative Analysis
In contrast to quantitative analysis where each cycle (log-linear
The complexity and rigor of the controls required depends on the
phase) of the PCR is analyzed, qualitative PCR is an end-point
degree of quantitation demanded for the reaction. There are
analysis after the PCR has reached plateau phase. Qualitative
three levels of qPCR quantitation and therefore three levels of
analysis is not as accurate as quantitative; however, it can provide
complexity for appropriately controlled reactions. The levels of
rapid and adequate information for some experiments. Qualita-
quantitation are: absolute quantitation, relative quantitation and
tive analysis is gel-based and provides a visual assessment of
non-quantitative or qualitative.
primer design, specificity and quality of the PCR amplification.
Absolute Quantitation
Summary
Absolute quantitation is the most rigorous of all quantitation
qPCR is a powerful and flexible technique, but the results achieved
levels. Absolute quantitation requires the addition of external
using this method are only valid if the appropriate controls have
standards in every reaction to determine the absolute amount of
been included in the experiment. The level of required controls
the target nucleic acid of interest. To eliminate potential differ-
depends on the level of quantitiation desired: absolute, relative
ences in quantitation due to annealing, the primer binding sites
or qualitative. Once that level of control has been identified,
of the external standards must be the same as those in the target
employing it when performing qPCR will contribute to the
sequence. The ideal external standard contains sequences that
success of the experiment.
are the same as the target sequence or which vary slightly from
the target sequence. Equivalent amplification efficiencies
between the target and the external standard are necessary for
absolute quantitation. Once a suitable construct or amplicon is
identified, a standard curve of external standard dilutions is
generated and used to determine the concentrations of unknown
target samples.

Our Innovation, Your Research — Shaping the Future of Life Science 17

 Optimizing qPCR
No matter whether your quantitation is to be absolute or relative, Figure 10. Analysis of Primers for Primer-Dimer Potential
the accuracy of qPCR depends on proper optimization of the
PCR and appropriate setting of the threshold value. While some A. Acceptable primers:
assays will not require complete optimization, there are a great
Optimizing qPCR

many delicate qPCR assays which require maximal selectivity and

sensitivity. For instance, pathogen detection or expression profiling
of rare mRNAs require high sensitivity. Assays such as SNP detection
or viral quantification require high specificity. Most challenging
are multiplex reactions, as these often require both sensitivity and
selectivity. By properly optimizing the conditions for the qPCR 3’-dimers > -2 kcal/mol
experiment, the researcher will be ensured of valid, reproducible Overall dimers > -6 kcal/mol
results with maximal specificity and sensitivity.
B. Unacceptable primers:

Guidelines for Optimizing Both

qPCR and qRT-PCR
Regardless of whether the target is DNA (qPCR) or RNA (qRT-PCR),
the following preliminary steps will aid in the optimization of the
reaction helping to ensure successful quantitation:
n Check primer design for primer-dimer potential 3’-dimers < -2 kcal/mol
n Optimize primer concentrations & extendable 3’-end
n Optimize probe concentration C. Unacceptable primer:
n Validate performance with a standard curve
n Prepare a melt curve
n Set the threshold value
Check Primer Design for Primer-Dimer Potential
The propensity of primers to hybridize to one another may lead
to primer extension during PCR and the formation of target- Overall dimer < -6 kcal/mol
independent products known as primer-dimers. This is especially
true for primers with complementarity at their 3’-ends. When Primer sequences were analyzed for their ability to form duplexes using Oligo
primer-dimer products are produced and amplified, the reaction 5.0 Primer Design Software. As described in the text, the primers shown in A
components are diverted from synthesis of the desired product, are not expected to form significant amounts of primer-dimer because they
thereby reducing assay efficiency and sensitivity. Therefore, only form weak duplexes. On the other hand, those in B hybridize too strongly
at the 3’-end and those in C hybridize too strongly overall.
primer-dimers are an issue in both probe-based and SYBR Green
dye-based detection. With SYBR Green dye-based detection,
Optimize Primer Concentrations
primer-dimers also affect assay specificity because the primers
will be detected along with the desired product. As a result, Satisfactory results for probe-based qPCR are often obtained
primers that are likely to form primer-dimers should be avoided, with final concentrations of both primers at 500 nM and the
most especially with SYBR Green dye-based detection. probe at 250 nM, especially if the PCR target is abundant and
maximum sensitivity is not required. Somewhat lower primer
To check the potential for primer-dimer formation, use primer levels, 200-400 nM, are usually better when using SYBR Green
design software to analyze duplex formation. Any 3’-terminal dye-based detection to minimize non-specific amplification.
dimers formed by either primer hybridizing with itself or with its Conduct a standard curve analysis, as described in the next
partner must be non-existent or very weak (DG ≥ –2.0 kcal, Fig. 10A). section. If detection is linear and efficiency is greater than 85%
Any primer with both a terminal DG ≥ –2.0 kcal and an extendable over the range of target expected in samples, it is not necessary
3’-end (5’-overlap, Fig. 10B) should be avoided. The strongest overall to optimize primer and probe concentrations.
dimer should be unstable as well (DG ≥ –6.0 kcal, Fig. 10C). To
avoid strong 3’-terminal dimers while maintaining specificity, choose
primers that have 2 G or C residues in the last 5 bases, 1 G or C
in the last 3 bases, and an A or T at the 3’-end (Fig. 10A).

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For maximum sensitivity, optimum primer concentrations must be 3. Aliquot 26 µL master mix into all wells in the PCR plate that
determined empirically. Primer concentrations are most efficiently contain primers (A1-E5).
optimized by testing various combinations in qPCR, as shown in
4. Mix thoroughly and transfer 18 µL from each of wells A1
the example below. Regardless of the detection chemistry used in
through E5 to wells A8 through E12.

Optimizing qPCR
the final assay, the best assay sensitivity will be obtained if primer
concentrations are optimized in the presence of SYBR Green. This 5. Add 2 µL template-containing DNA (10-50 ng genomic DNA or
allows detection of primer-dimer and other non-specific products, 0.1-1 ng plasmid) or RNA (10-100 ng total RNA or 0.5-10 ng
and helps the user to screen out reactions with multiple products mRNA) to one set of reactions (columns 1-5) and 2 µL water to
(step 8, next section). Alternatively, if maximum sensitivity is not the other (columns 8-12).
a concern, the corresponding probe may be included in reactions 6. Perform thermal cycling:
at 250 nM.
Time Time
Primer Optimization Example Number
Temperature for for
1. Prepare and dispense diluted primers of Cycles
qPCR qRT-PCR
a. Prepare 60 μL of 8 μM working solutions of both forward
Reverse
(fwd) and reverse (rev) primers in the first tubes of 2 1 45 °C 0 min 15-30 min
transcription
separate 8-tube strips.
b. Dispense 30 μL of water into tubes 2-5. Denature 1 94 °C 3 min 3 min
c. Transfer 30 μL of the 8 μM primer solution from tube 1 into Denature 40 94 °C 15 sec 15 sec
tube 2. Mix thoroughly by pipetting up and down at least
Anneal, extend,
5 times.
and read 60 °C 1 min 1 min
d. Repeat transfer and mixing from tube 2 to 3, 3 to 4, and
fluorescence
4 to 5.
e. Using a multichannel pipettor, transfer 5 μL from the strip- Dissociation/
1 * * *
tubes containing diluted fwd primer into the first 5 wells melting curve
down columns 1-5 of a 96-well PCR plate. After adding *See manufacturer’s instructions for the real-time thermal cycler used.
reverse primer, PCR mix, and template (below), final
7. Evaluate fluorescence plots (DRn) for reactions containing
concentrations of forward primer will be 1000, 500, 250,
target nucleic acid (columns 1-5). Primer combinations with the
125, and 62.5 nM.
lowest Ct and the highest fluorescence will give the most
f. Similarly, transfer 5 μL from the strip-tubes containing diluted
sensitive and reproducible assays.
rev primer into the first 5 wells across rows A-E. After adding
PCR mix and template (below), final concentrations of 8. Evaluate dissociation/melting curves. Primer combinations
reverse primer will be 1000, 500, 250, 125, and 62.5 nM. with single, sharp peaks in the presence of target nucleic acid
(columns 1-5) and nothing detected in the corresponding
2. Prepare qPCR or qRT-PCR master mix (for 52 × 20 µL reactions):
no-template control (columns 8-12) will give the most sensitive
qPCR and reproducible assays. If all primer combinations give some
product in the absence of template, and this no-template
Reagent Catalog Number Volume
product melts at a lower temperature than that with template,
Water W1754 155 µL select the combination that gives the least amount of lower-
SYBR Green JumpStart™ Taq melting no-template product. The latter is likely primer-dimer.
S9939* 520 µL Detection can be avoided, or at least minimized, by adding a
ReadyMix™
15 second melting step approximately 3 °C below the melting
Reference dye** R4526* 1.0 µL
temperature of the desired PCR product during which fluores-
qRT-PCR cence is measured after the annealing/extension step in
each cycle.
Reagent Catalog Number Volume
Water W1754 123.8 µL Optimize Probe Concentration
For maximum sensitivity, 250 nM probe may be used in all assays.
SYBR Green JumpStart Taq ™
S9939* 520 µL However, if maximum sensitivity is not required, lower levels of
ReadyMix™
probe may suffice, thereby reducing the assay cost. To optimize
Reference dye** R4526* 1.0 µL probe concentration, test the probe at several levels from 50 to
40 U/µL RNase inhibitor R2520 26 µL 250 nM final concentrations in PCR with optimized levels of
200 U/µL MMLV reverse primers and the lowest level of target nucleic acid expected. The
M1427 5.2 µL lowest level of probe that allows acceptable detection (Ct ≤ 30
transcriptase
for best reproducibility) may be used.
* S9939 and R4526 are components of Catalog Number S4438.
** Use 10 × more for ABI 7700; replace with FITC for BioRad iCycler.

Our Innovation, Your Research — Shaping the Future of Life Science 19

 Optimizing qPCR
Validate Performance with a Standard Curve Figure 11. Use of Standard Curves to Evaluate
A standard curve, generated by performing qPCR with a serial qPCR Optimization
dilution of template, is an excellent tool to test assay efficiency, A.
precision, sensitivity, and working range. Prepare at least three, but 40
Optimizing qPCR

preferably five or more, 4- to 10-fold serial dilutions from a DNA

or RNA sample that contains the PCR target. Plan this dilution 38
series to extend past both the highest and lowest levels of target
expected in test samples. Conduct qPCR with all dilutions and 36
with a no-template control, using previously optimized primer and
34
probe concentrations. With SYBR Green dye-based detection,
also include a melt curve test at the end of thermo­cycling. The 32

Ct
software for most real-time qPCR instruments can be set up
to prepare a standard curve and to calculate efficiency (see the 30
user guide for the instrument being used). If this feature is not
28
available, prepare a plot of Ct versus the log of nucleic acid input
level and perform a linear regression. Calculate the reaction 26
efficiency from the slope of the line using the equation:
24
Efficiency = 10(–1/slope)-1
-3 -2 -1 0
The correlation coefficient of the line, R2, is a measure of how Log of DNA dilution
well the data fits the model and how well the data fits on a straight B.
line, and is influenced by pipetting accuracy and by the range of
35
the assay. If R2 is ≤ 0.985, the assay may not give reliable results.
If one or more points at the lowest levels of input nucleic acid are
shifted away from the linear region of the plot, it is likely that the 30
level exceeds assay sensitivity (Fig. 11A). To improve sensitivity,
optimize primer concentrations or design different primers. 25
Similarly, if one or more points at the highest levels of input
nucleic acid are shifted away from the linear region of the plot, 20
Ct

it is likely that the reaction is saturated and that the level of

target exceeds the useful assay range (Fig. 11B). To address this
15
situation, add less or dilute the sample nucleic acid. Alternatively,
if several random points are above or below the line, pipetting
accuracy may be a problem. Verify that the pipette tips fit the 10
pipettor properly and that the volume dispensed is reproducible.
If the PCR is 100% efficient, the amount of PCR product will 5
double with each cycle and the slope of the standard curve will -5 -4 -3 -2 -1 0
be –3.33 (100 = 100% = 10(–1/–3.33)-1). A slope between –3.9 and Log of DNA dilution
–3.0 (80-110% efficiency) is generally acceptable. Calculated
levels of target input may not be accurate if the reaction is not A. Assay not linear at low levels of input nucleic acid. B. Assay not linear at
high levels of input nucleic acid.
efficient. To improve efficiency, optimize primer concentrations
or design alternative primers.

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Prepare a Melt Curve Setting the Threshold Value

Since SYBR Green binding dye is a non-specific dye that will detect There are several methods that can be used to calculate the
any double-stranded DNA, it is important to verify that the PCR threshold value. The critical factors for determining the level of
produces only the desired product. This can often be detected when the threshold are: (a) the fluorescence value must be statistically

Optimizing qPCR
PCR efficiencies are larger than 120%. Melt, or dissociation, curve higher than the background signal, (b) the samples must all be
analysis can also be used to determine the number and approxi- measured in the exponential phase of amplification and (c) the
mate size of products. An assay with high specificity will give efficiency of amplification must be identical for all samples.
a single peak at a high temperature (> 80 °C) in all reactions
The fluorescence value must be statistically higher than the
and nothing, or very little, detected in the no-template controls
background signal to ensure that real data are collected. Most
(Fig. 12A). If the melting curve has more than one major peak,
instruments automatically calculate a threshold level of fluores-
as in Figures 12B and 12C, the identities of the products should
cence signal by determining the baseline (background) average
be determined by fractionating them on an ethidum bromide-
signal and setting a threshold 10-fold higher than the baseline
stained agarose gel. As shown in Figures 12E and 12F, reactions B
average signal.
and C contain excessive amounts of primer-dimer or other non-
specific products. Lowering the primer concentrations will often Setting a manual threshold is best accomplished using a log
reduce the amount of non-specific products. If non-specific signal plot, as the exponential part of the curve shows clearly
products are still detected in significant amounts with low primer as a linear trace.
levels, redesign the primers.

Figure 12. Evaluation of Melt Curves

A. B. C.

D. E. F.

Melt, or dissociation, curves showing a sharp peak of specific product at > 80°C, very little non-specific product at lower temperatures (A), or significant amounts of
non-specific, lower melting product (B&C). D-F show PCR products from A-C, respectively, fractionated on ethidium bromide-stained 2% agarose gels.

Our Innovation, Your Research — Shaping the Future of Life Science 21

 Optimizing qPCR
Additional Guidelines for If a Bioanalyzer is not available, 1-2 µg of total RNA may be
evaluated by ethidium bromide-stained agarose gel electrophore-
Quantitative Reverse Transcription sis to verify that the RNA appears reasonably intact. For good
quality total RNA, the two largest rRNAs should appear as
PCR (qRT-PCR)
Optimizing qPCR

discrete bands at approximately 5 kb and 2 kb and the upper

When performing qRT-PCR, it is not only important to consider band should have approximately twice the intensity of the lower.
the guidelines for standard qPCR, but for optimum qRT-PCR The mRNA should appear as a light smear, mostly between the
results, the following points should be addressed as well: two rRNA bands, as in lanes 1 and 2 in Figure 14. The RNA in
n Verify RNA quality lanes 3 and 4 of Figure 14 are partially degraded.

n Confirm that primers span or flank long introns Figure 14. Evaluation of RNA Integrity by Agarose
n Conduct no-reverse transcriptase (no-RT) controls Gel Analysis
n Optimize reverse transcription 1 2 3 4

For additional information, please see Appendix 2: How to

Optimize your Quantitative Real-Time RT-PCR (qRT-PCR).
Verify RNA Quality
The quality of total RNA is most readily assessed by capillary
electrophoresis with an Agilent 2100 bioanalyzer. The instrument 28S rRNA
software evaluates the proportion of RNA detected before,
between, and after the rRNA peaks to determine a relative 18S rRNA
integrity number (RIN) for the RNA sample analyzed. Perfectly
intact RNA has a RIN of 10 whereas completely degraded RNA
has a RIN of 1. Whether or not partially degraded RNA (RIN < 9)
will give satisfactory results in qRT-PCR depends upon the level of
sensitivity required as well as the RT-PCR strategy. For example, an Samples of total RNA (2 μg) were fractionated on a 1% agarose gel in TBE
buffer and stained with ethidium bromide.
RT-PCR strategy that uses a two-step Oligo-dT to prime reverse
transcription and PCR primers near the 5’-end of a long cDNA
Confirm that Primers Span or Flank Long Introns
will require much higher integrity than a strategy that uses
one-step RT-PCR with gene-specific primers. In addition, higher While most DNA is eliminated during RNA purification, no procedure
integrity will be required to detect a rare mRNA compared to an removes 100% of the DNA. Because PCR can amplify even a
abundant mRNA. Therefore, the correlation between RIN and single molecule of DNA, RT-PCR can amplify contaminating DNA
qRT-PCR success must be determined empirically for each assay. as well as RNA. If the target mRNA is fairly abundant (hundreds or
thousands of copies per cell), DNA amplification will be negligible
By way of example, the RNA samples with Bioanalyzer traces in comparison to the products from the RNA. If, however, the
shown in Figure 13, gave quantifiable difference for several rare target mRNA is less than 100 copies/cell, DNA amplification can
mRNAs when using a one-step qRT-PCR using gene-specific lead to erroneously high estimates of mRNA levels. To avoid DNA
primers. This same RNA template was quantified to have relative amplification during RT-PCR, use primers that either flank an
mRNA amounts varying by four-fold when using two-step qRT-PCR intron that is not present in the mRNA sequence or that span an
with Oligo-dT to prime RT. The latter difference was shown to exon-exon junction (Fig. 15). If both genomic and cDNA sequences
correlate to RIN, as mRNAs were detected up to 2 cycles later in for the target mRNA are publicly available, intron positions can be
sample 11 (RIN = 9.8) than in sample 9 (RIN = 7.0). identified by performing a BLAST search with the cDNA sequence
against the genomic database for the target organism (Fig. 16).
Figure 13. Evaluation of RNA Integrity with the DNA sequences with short intervening sequences (~1 kb) may
Agilent Bioanalyzer 2100
be amplified in RT-PCR (e.g. Fig. 17A). For example, Intron 1 in
Electropherogram Virtual Gel Figure 16 is long enough (~ 6.5 kb) to preclude amplification of
the genomic DNA, while all other introns are short (< 1 kb) and
likely will be amplified during RT-PCR. The example in these two
sample 9
RIN = 9.8
figures illustrates that, if possible, primers should either span
exon-exon junctions, flank a long (several kb) intron, or flank
multiple small introns.
sample 11
If the gene of interest has no introns, if the intron positions are
RIN = 7.0
unknown, or if there are no suitable primers that span or flank
introns, it may be necessary to digest input RNA with an RNase-
free or amplification-grade DNase I. Conduct no-RT controls to
Samples of total RNA preparations (~150 ng) were fractionated on a RNA 6000 determine whether or not digestion with DNase I is needed.
Nanochip and integrity was evaluated before use in qRT-PCR.

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 INNOVATION @ WORK

Figure 15. Illustration of Intron-Spanning (A) and Figure 17. Evaluation of No-RT Controls
Intron-Flanking (B) Primers for RT-PCR
A. B.
A. Primers Span Intron mw + – + – +/– RT
P2

Optimizing qPCR
P2
DNA:
P1 P1 2,000

P2
1,500

mRNA:
P1 1,000

B. Primers Flank Intron 750

P4
DNA: 500
P3

300 294
Introns are in red, exons are in green. Primers P1 & 2 span an intron and primers
P3 & 4 flank an intron. Note that primers P1 & 2 are only partly complimentary 150 191
to the gDNA strand and will not generate a PCR product from DNA unless the 50
annealing temperature is extremely low. P3 & 4 may generate a longer PCR
product from DNA if the intron is short (~1 kb), but not if it is sufficiently long
(several kb). RT-PCR products produced in the presence (+) or absence (-) of RT enzyme
were fractionated on an ethidium bromide-stained 2% agarose gel in TBE.
Figure 16. BLAST Alignment of cDNA Sequence with Primers for the mRNA target in (A) flank a 1 kb intron. Note the 1.5 kb band
Genomic DNA Sequence in the no RT control. The mRNA target in (B) aligns with several genes, at least
one of which is a pseudogene that lacks the intron between the primers used
for RT-PCR. As such, the no RT control gives a larger yield than when reverse
transcriptase is added.

Conduct No-Reverse Transcriptase (No-RT) Controls

Regardless of whether primers span or flank introns, the specificity
of qRT-PCR assays should be tested in reactions without reverse
transcriptase (no-RT control) to evaluate the specificity of DNA
amplification. As mentioned above, DNA sequences with short
introns (≤ 1 kb) may be amplified in RT-PCR. Many genes have
additional copies, or pseudogenes, that lack one or more introns
(Fig. 17B). As a result, qRT-PCR assays should be tested for
potential DNA-only amplicons by performing reactions that
contain RT, the same RNA, but no RT enzyme. DNA amplification
is not a problem if the Ct values for no-RT reactions are at least
5 cycles greater (32-fold less) than those for reactions with RT.
However, if there are fewer than 5 cycles between Ct values for
reactions with and without RT, DNA amplification may skew
The complete cDNA sequence for rat p53 from Genbank (accession number
NM_030989) was used in a megaBLAST search for identical sequences in the rat
attempts at mRNA quantitation.
genome (http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html). The alignment In cases where DNA amplicons contribute significantly, one should
on chromosome 10 is shown; each tick mark on the scale represents 1 kb.
digest the RNA with an RNase-free or amplification grade DNase I
before qRT-PCR to allow reliable mRNA quantitation. Note that
on-column DNase digestion, which is commercially available in
several RNA purification kits, is less effective at eliminating DNA
than digestion in solution after eluting RNA from the column.
As a result, on-column (OC) DNase digestion may or may not be
sufficient for qRT-PCR (Fig. 18, see +OC DNAse -RT samples versus
post prep DNAse -RT). No-RT controls should be conducted with
DNase-digested RNA to verify that the digestion was successful
and sufficient. In the example shown in Figure 17, OC DNase
digestion is sufficient to reliably detect the target mRNA. It would
not be sufficient to reliably quantitate a less abundant mRNA, if
the samples contained less of the mRNA shown, or if greater
sensitivity was required.

Our Innovation, Your Research — Shaping the Future of Life Science 23

 Optimizing qPCR
Note that different types of cells and tissues, as well as different The temperature used for RT reactions may affect specificity,
growth conditions, produce significantly different levels of especially with gene-specific primers. Primers that can form a
specific mRNAs. In addition, different RNA purification methods strong 3’-duplex will hybridize more readily at lower tempera-
give different levels of contaminating DNA. As a result, reactions tures. Since RT enzymes can extend a DNA primer on a DNA
Optimizing qPCR

with and without RT should be performed at least once with each template, primer-dimer formation may start during the RT step.
new starting material and RNA preparation method. Increasing RT incubation temperature to the highest temperature
at which the enzyme is fully active or using a high-temperature
Figure 18. Comparison of On-Column DNase Digestion enzyme may reduce the amount of primer-dimer. For example,
(OC) with Post-Preparation DNase Digestion the primers used in Figure 18 gave significantly less non-specific
5 product in one-step qRT-PCR when RT was performed with MMLV-RT
(Moloney Murine Leukemia Virus-Reverse Transcriptase) at 45 °C
4
(Fig. 19B) than when the reaction was performed at 37 °C
(Fig. 19A). Similarly, performing two-step RT-PCR with a non-
Fluorescence (dRn)

specific primer for RT and hot-start Taq polymerase for qPCR may
3

+/– DNase give less primer-dimer (Fig. 19D) than one-step qRT-PCR with
+ RT gene specific primers that can form a 3’-duplex (Fig. 19B).
2 
– DNase
– RT The amount of RT enzyme per reaction can also affect qRT-PCR
+ OC DNase
– RT results. As shown in Figure 18, one-step reactions with 2 units of
1 MMLV-RT (Fig. 19C) gave better specificity than reactions with
+ post-prep DNase 20 units (Fig. 19B). Superscript™ III, an RNaseH– deletion of
– RT
0 MMLV-RT, and Omniscript from Qiagen gave results similar to
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 those shown in Figure 18 (data not shown). Two-step RT-PCR
Cycles
with Oligo-dT or random primers for RT often gives greater
specificity than does one-step RT-PCR (Fig. 19D). This could be
Total RNA was prepared from 30 mg pieces of mouse liver with either the attributed to the fact that the gene-specific primers are not
Sigma GenElute™ Total RNA Kit or the Qiagen RNeasy Mini Kit according to the
present to form non-specific products during the low tempera-
manufacturers’ instructions. Two RNA samples were prepared with the respec-
tive manufacturer’s on-column DNase product and two were prepared without ture RT reaction. Higher levels of RT may give better results in
DNase digestion. After purification, aliquots of the four RNA samples prepared two-step reactions, but because the RT enzyme can interfere with
without on-column DNase were digested with Sigma’s Amplification Grade Taq activity, the amount of RT product transferred to qPCR should
DNase I according to the manufacturer’s instructions. Equal proportions of all be limited to no more than 10% of the final reaction volume.
were used in one-step qRT-PCR. Fluorescence plots for two of the RNA samples
are shown. Similar results were obtained with both manufacturers’ products.

Optimize Reverse Transcription

The choice of primers used to initiate reverse transcription can
greatly affect qRT-PCR results. For one-step qRT-PCR, gene-
specific primers must be used. When performing a two-step
assay, a reverse gene-specific primer, Oligo-dT, random hexamers,
nonamers, decamers, or dodecamers may be used. Gene-specific
primers require separate reactions for each target RNA. These
separate reactions may have very different efficiencies, thus
complicating comparisons between RNA levels. On the other
hand, with a gene-specific primer, all of the RT product will
encode the gene of interest and may allow quantitation of very
low abundance mRNAs not detected using non-specific RT primers.
Give these complications, the choice of RT priming should be
carefully considered. To avoid the potentially high inter-assay
variations in RT that can occur with gene-specific primers,
non-specific primers may be used to generate a pool of cDNA.
This would be followed by separate qPCR assays for each target
performed with aliquots from the cDNA pool. If all qPCR targets
are near the 3’-end of polyadenylated mRNAs, oligo-dT is a good
choice for primer. On the other hand, if the qPCR targets are
more than a few kilobases from the 3’-end or if the RNA is not
polyadenylated, random hexamers, octamers, nonamers, or
decamers will give better detection. If the location of qPCR targets
or the polyadenylation level of RNAs varies, a mixture of Oligo-dT
and random oligomers will give the best results.

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Figure 19. Optimization of RT

A. B.

Optimizing qPCR
C. D.

Melt curves of RT-PCR products produced with one-step (A-C) or two-step (D) qRT-PCR. Reactions (A-C) each contained 10 µL of SYBR Green JumpStart Taq
ReadyMix, 0.02 µL of Reference Dye, both gene-specific primers at 0.4 µM, and 10 ng human total RNA in a final volume of 20 µL. Gene-specific primers were
5’-CGGGCTTCAACGCAGACTA-3’ and 5’-CTGGTCGAGATGGCAGTGA-3’ for c-fos (Accession NM_005252). Reactions (A&B) also contained 20 units of MMLV- RT,
whereas reaction (C) contained 2 units. Reaction A was incubated at 37 °C for 30 min before qPCR, whereas (B&C) were incubated at 45 °C for 30 min before qPCR.
In (D), the RT reaction contained 1× MMLV buffer, 0.5 mM dNTPs, 1 µM Oligo-dT, 0.8 units/µL RNase inhibitor, 200 units MMLV-RT, and 10 ng human total RNA in a
final volume of 20 µL. The reaction was incubated at 25 °C for 10 min, 37 °C for 50 min, and 80 °C for 10 min. 2 µL of the RT reaction product was added to qPCR
containing 10 µL of SYBR Green JumpStart Taq ReadyMix, 0.02 µL of Reference Dye, and both gene-specific primers at 0.4 µM as for the one-step reactions (A-C).
All qPCR reactions were incubated at 94 °C for 3 min to denature, then for 40 cycles of 94 °C for 15 sec and 60 °C for 1 min.

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 Optimizing qPCR
Additional Optimization for Optimize Mg2+ Concentration
Magnesium plays several roles in PCR. It is a required divalent
Multiplex Reactions cationic counter-ion for dNTPs and a co-factor for all polymerases.
Successful multiplex qPCR, in which more than one target is Divalent cations strongly affect DNA double-strand hybridization,
Optimizing qPCR

quantitated in a single reaction, often require additional optimiza- and increasing magnesium raises the stability, or melting tempera-
tion. One simple consideration is to minimize the spectral separation ture, of a DNA duplex. It follows that high magnesium levels
of the multiple emissions. This facilitates signal isolation and data increase the affinity of primers toward hybridization, including mis-
analysis. As a result, fluorophores with narrow, well-resolved priming events and primer-primer interactions. The mis-primed
bandwidths are useful for multiplex applications. Appendix 1 DNA duplexes become substrates for the DNA polymerase, in
contains Traits of Common Fluorophores to aid in the selection of effect creating side products and sapping PCR efficiency. Salts,
fluorophores. For multiplex reactions, it is also recommended to such as KCl, will also change DNA duplex Tm, but the effect is
optimize the following: less drastic for these monovalent cations.
n Check primer design PCR requires a minimal amount of magnesium, and both efficiency
n Optimize primer concentartions and product Tm change as the cation concentration increases.
These effects are magnified when one attempts to perform
n Optimize Mg2+ concentration multiplex PCR. Running multiple reactions concurrently introduces
Check Primer Design competition for reagents and exacerbates any non-optimal conditions
As for single-target reactions, multiplex qPCR will give the best results creating major changes in PCR efficiency. Figure 20 demonstrates
if all primers in the reaction have similar melting temperatures this point. The efficiency curves for two primer/probe targets were
(Tm difference ≤ 2 °C) and none can form strong 3’-duplexes performed individually and then in multiplex. The graph shows
(DG ≥ –2.0 kcal). For more information, see the section Check that while the individual reactions (dark blue and green lines)
Primer Design for Primer Potential on page 18. Individual reaction give relatively similar efficiencies and sensitivities (y-axis values)
optimizations should be performed as well as optimization with running the reactions together dramatically changes the sensitivity
several or all primer combinations. It is very often the case that and efficiency of the later reaction.
individual primers work singly, but when combined in multiplex
the primers cross-react or otherwise alter reaction specificity
and efficiency.
Optimize Primer Concentrations
If one target in a multiplex reaction is significantly more abundant
than the other(s) or if one primer pair gives a much lower Ct or
higher DR (the amount of fluorescence in the no-template control)
than the other(s), amplification of that target may dominate the
reaction, using up reactants before other targets are detectable.
Adjusting the levels of primers may allow a more balanced
amplification of all targets. To determine if such adjustments
will be beneficial, prepare standard curves that cover the range
of targets expected for each primer pair alone (singleplex) and
with all primers combined (multiplex). There is no need to modify
primer levels if multiplex and singleplex reactions give similar results.
On the other hand, optimizing primer concentrations will likely
improve results if sensitivity is unacceptable in multiplex reactions.
Decrease primer concentrations for those primer pairs that give
low Ct values and/or increase concentrations for those that give
high Ct values, within the range of 50-500 nM.

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Figure 20. Singleplex Reaction vs. Duplex Reaction

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Optimizing qPCR
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Our Innovation, Your Research — Shaping the Future of Life Science 27

 qPCR Reagent Selection Table
Use the following table to choose the most appropriate reagent High-throughput mixes include the reference dye in the ReadyMix
for your needs. The table is read horizontally to select the thermal to eliminate an extra pipetting step. The Quantitative RT-PCR
cycler format. Products are compatible with tube, plate, or reagents are noted in gray. These are for use when RNA is used
capillary-based instruments. The table is read vertically to narrow as the starting template.
qPCR Reagent Selection Table

the reagent to SYBR Green dye or probe-based formulations.

qPCR Reagent Selection Table

Plate/Tube Instruments Glass Capillary Instruments
Catalog Catalog
Number Product Name Package Size Number Product Name Package Size
S4438 SYBR Green JumpStart Taq 100 Reactions S1816 SYBR Green JumpStart Taq 20 Reactions
ReadyMix for Quantitative PCR 500 Reactions ReadyMix for Quantitative PCR, 100 Reactions
Capillary Formulation 400 Reactions
SYBR Green based qPCR

S5193 SYBR Green JumpStart Taq 20 Reactions S5193 SYBR Green JumpStart Taq 20 Reactions
ReadyMix without MgCl2 100 Reactions ReadyMix without MgCl2 100 Reactions
(with separate tube of MgCl2) 400 Reactions (with separate tube of MgCl2) 400 Reactions
S9194 SYBR Green JumpStart Taq 20 Reactions
ReadyMix for High-Throughput 400 Reactions
Quantitative PCR (with internal 2000 Reactions
reference dye)
QR0100 SYBR Green Quantitative 1 Kit QR0100 SYBR Green Quantitative 1 Kit
RT-PCR Kit (100 Reactions) RT-PCR Kit (100 Reactions)
Catalog Catalog
Number Product Name Package Size Number Product Name Package Size
D7440 JumpStart Taq ReadyMix for 100 Reactions D9191 JumpStart Taq ReadyMix 20 Reactions
Quantitative PCR 400 Reactions with dUTP 100 Reactions
400 Reactions
Probe based qPCR

D9191 JumpStart Taq ReadyMix 20 Reactions

with dUTP 100 Reactions
400 Reactions
D6442 JumpStart Taq ReadyMix for 20 Reactions
High-Throughput Quantitative 400 Reactions
PCR (with internal 2000 Reactions
reference dye)
QR0200 Quantitive RT-PCR ReadyMix 1 Kit QR0200 Quantitive RT-PCR ReadyMix 1 Kit
(100 Reactions) (100 Reactions)
Package sizes based on 50 µL reactions

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Troubleshooting
Fluorescence Issues
No, or low, fluorescence in both the test sample and in the positive control with the correct PCR
Number 1
product on the gel

Troubleshooting
SYBR Green Dye-Based Detection Probe Detection
Possible cause Bad SYBR Green binding dye High-background fluorescence Degraded probe
Compare fluorescence of the SYBR Check the raw fluorescence Digest 5 fmoles of probe with
Green binding dye mix ± 1 µg DNA. (multicomponent plot). Fluorescence DNase I. Fluorescence should be at
Diagnostic test Fluorescence should be at least should increase at least 10,000 units least 10,000 units greater than
10,000 units higher with DNA between cycles 1 & 40 without DNase digestion
added than without
Purchase new SYBR Green binding Purchase a new probe Purchase a new probe
Solution dye or a new qPCR mix with SYBR
Green binding dye
Number 2 Low fluorescence from test sample, but the positive control has good fluorescence
Possible cause Fluorescence quenching
Diagnostic test Positive control gives good fluorescence
Solution Purify the input nucleic acid
Number 3 Declining or hooked fluorescence plots
As PCR product accumulates, the complimentary strand competes with the primer and/or probe for annealing
Possible cause
to template
Diagnostic test See figure
Solution Ignore it if the Ct is not affected

Figure 21. Hooked Fluorescence Plots

Our Innovation, Your Research — Shaping the Future of Life Science 29

 Troubleshooting
Fluorescence Issues
Number 4 The fluorescence plots suddenly spike upwards
Possible cause Bad reference dye
Troubleshooting

Disable reference dye normalization or look at the results for DR (level of fluorescence in the no-template control)
Diagnostic test instead of DRn (the difference of the reporter fluorescence in the sample and that in the no template control). The
plots should become smooth if fluorescence is not normalized to an inactive reference dye
Solution Purchase a new reference dye. Always protect dye from light during storage
Number 5 No amplification results from a sample known to contain target and the positive control does amplify
Possible cause Inhibition in the test sample is likely
Test amplification with a diluted sample. Spike the reaction with a low level of exogenous target and test for
Diagnostic test
amplification of the exogenous target
Solution Try adding BSA to 0.3% in the PCR or purify the input nucleic acid

Dissociation/Melting Curves
Number 6 There are multiple peaks in the dissociation plot/melt curve
Possible cause The Mg2+ concentration in the reaction is too high or the annealing temperature is too low
Fractionate the PCR product on an ethidium bromide-stained agarose gel or use the Bioanalyzer to verify
Diagnostic test
multiple products
Titrate the Mg2+ to determine the optimum concentration. Perform annealing temperature gradient to select the
Solution
optimum annealing temperature
Number 7 There is a broad peak at a lower Tm than the desired product, especially in low or no-template reactions
Possible cause Primer-dimer, generated from primers that anneal at their 3’-ends, extend, and then amplify
Fractionate the PCR product on an ethidium bromide-stained agarose gel or use the Bioanalyzer. The primer-dimer
Diagnostic test
will appear as a diffuse band at ≤ 50 bp
Try lower primer concentrations. With RT-PCR, try using less RT enzyme and use a 2-step and/or a higher
Solution
incubation temperature for the RT step
Number 8 Multiple Tm peaks
Single product on gel Multiple products on gel
Localized AT or GC-rich regions or short repeats in Multiple targets in the source material
Possible cause
PCR product
Check sequence of amplicon for AT or GC-rich regions BLAST primer sequences against the sequence of the
Diagnostic test
or repeats source organism to verify single target
This is not a problem if the gel analysis shows that all Design unique primers
Solution
product is specific. Continue to use the primers

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Standard Curve
Number 9 PCR efficiency < 80%
Suboptimal PCR conditions (See example. This illustrates Poorly designed primers
Possible cause

Troubleshooting
low Taq activity)
Prepare or purchase fresh PCR mix Check the primer design. Test the PCR mix with a set
Diagnostic test of primers known to work well and a positive
control template
Solution Prepare or purchase fresh PCR mix Design new primers
Number 10 PCR efficiency is greater than 120%
Possible cause Excessive primer-dimer Pipetting is inaccurate Inhibition
Evaluate dissociation plot as shown Test pipette calibration See Problem Number 4: The
Diagnostic test in the figure below fluorescence plots suddenly spike
upward
Try lower primer concentrations. If the pipettes are inaccurate, get See Problem #4: The fluorescence
With RT-PCR, try using less RT them re-calibrated. If the pipettes plots suddenly spike upward
enzyme, doing 2-step and/or using are properly calibrated, practice
Solution a higher incubation temperature for pipetting accurately
the RT step

Our Innovation, Your Research — Shaping the Future of Life Science 31

 Troubleshooting
Standard Curve
Number 11 The standard curve is not linear with low levels of sample input
Possible cause Exceeded assay sensitivity
Troubleshooting

Diagnostic test Ct > 30; qPCR replicates highly variable

Solution Optimize primer concentrations

Figure 22. Standard Curve that is not Linear with Low Levels of Sample Input

40

38

36

34
Ct

32

30

28

26

24
-3 -2 -1 0
Log of DNA dilution

Number 12 The standard curve is not linear with high levels of sample input
Possible cause Exceeded assay capacity
Diagnostic test Low Ct (< 12-15, depending on instrument)
Solution Use less input nucleic acid

Figure 23. Standard Curve that is not Linear with High Levels of Sample Input

35

30

25
Ct

20

15

10

5
-5 -4 -3 -2 -1 0
Log of DNA dilution

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 INNOVATION @ WORK

qRT-PCR Specific
Number 13 Product is detected in the no-RT reaction with primers that flank an intron
The intron is short enough that the primers amplify Pseudogenes are present that lack introns
Possible cause

Troubleshooting
(~1.4 kb in example)
Fractionate the PCR product on an ethidium bromide- Fractionate PCR product on ethidium bromide-stained
Diagnostic test stained agarose gel. No-RT product will migrate more agarose gel. No-RT product will migrate the same as
slowly than +RT product as in gel photograph shown +RT on gel
Solution Re-design the primers to flank or span a longer intron or multiple introns

Multiplex
Number 14 One, or more, primer/probe set does not work well in a multiplex reaction
Possible cause The target is less abundant or the primers are less efficient
Diagnostic test Conduct reactions with individual primer/probe sets to see if they work adequately in the absence of competition
Limit the level of primers that dominate the reaction and give higher signal. Determine the level that reduces DRn
Solution
without increasing Ct significantly

Sigma Product Specific

Number 15 Sigma’s JumpStart Taq doesn’t work as well as Qiagen’s HotStarTaq
Possible cause Reactions were denatured at 95 °C for 15 min before cycling
Diagnostic test Check the thermocycling profile
Solution 3 min at 94 °C is sufficient to activate JumpStart Taq; longer incubations will partially inactivate
Number 16 Sigma’s ReadyMix doesn’t work, but competitors do
Possible cause REDTaq ReadyMix was used for real-time qPCR
Diagnostic test Check color of qPCR mix
Solution Use clear mixes for real-time qPCR. Red dye will interfere with fluorescence detection for most instruments

Our Innovation, Your Research — Shaping the Future of Life Science 33

 Appendix 1
Traits of Commonly Used Fluorophores
Abmax Extinction Coefficient Emmax
Dye
(nm) (l mole–1 cm–1) (nm)
Appendix 1

Acridin 362 11,000 462

AMCA 353 19,000 442
BODIPY® FL-Br2 531 75,000 545
BODIPY 530/550
®
534 77,000 554
BODIPY® TMR 544 56,000 570
BODIPY® 558/568 558 97,000 569
BODIPY 564/570
®
563 142,000 569
BODIPY® 576/589 575 83,000 588
BODIPY® 581/591 581 136,000 591
BODIPY TR ®
588 68,000 616
BODIPY® 630/650 625 101,000 640
BODIPY® 650/665 646 102,000 660
Cascade Blue ®
396 29,000 410
Cy®2 489 150,000 506
Cy®3 552 150,000 570
Cy 3.5
®
581 150,000 596
Cy®5 643 250,000 667
Cy 5.5
®
675 250,000 694
Cy®7 743 250,000 767
DABCYL 453 32,000 None
EDANS 335 5,900 493
Eosin 521 95,000 544
Erythrosin 529 90,000 553
Fluorescein 492 78,000 520
6-FAM™ 494 83,000 518
TET™ 521 ­— 536
JOE ™
520 71,000 548
HEX™ 535 ­— 556
LightCycler® 640 625 110,000 640
LightCycler® 705 685 ­— 705
NBD 466 22,000 535
Oregon Green® 488 492 88,000 517
Oregon Green 500 ®
499 78,000 519
Oregon Green® 514 506 85,000 526
Rhodamine 6G™ 524 102,000 550
Rhodamine Green™ 504 78,000 532
Rhodamine Red™ 560 129,000 580
Rhodol Green™ 496 63,000 523
TAMRA ™
565 91,000 580
ROX™ 585 82,000 605
Texas Red™ 583 116,000 603
NED ™
546 not available 575
VIC® 538 not available 554
Yakima Yellow ™
526 84,000 448

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Appendix 2
How to Optimise Your Quantitative or in the first extraction buffer containing guanidine isocyanate,
provided in the extraction kits, and others freeze it in liquid
Real-Time RT-PCR (qRT-PCR) nitrogen. The extracted analyte (either total RNA or poly-
Michael W. Pfaffl, Physiology Weihenstephan, Technical University of Munich adenylated mRNA) as well as the RNA isolated using columns or a

Appendix 2
(TUM), Weihenstephaner Berg 3, 85354 Freising Weihenstephan, Germany liquid-liquid extraction, could result in varying RNA qualities and
http://optimisation.gene-quantification.info quantities. For example RNA extracted from collagen rich or
adipose tissues often has a lower total RNA yield, is of lesser qual-
Introduction ity, and contains partly degraded RNA sub-fractions. Particular
RNA extraction techniques can work more effectively in one
To get very reliable and quantitative real-time RT-PCR results, the specific tissue type compared with another one and result in up
applied mRNA quantification assay and working procedure to 10-fold variations in total RNA yield per extracted tissue mass
should be highly optimised. The efficacy of kinetic RT-PCR is and as a result, on the following real-time RT-PCR gene expres-
measured by its specificity, low background fluorescence, steep sion analysis as well. A lot of total RNA preparations are contami-
fluorescence increase and high amplification efficiency, and nated with genomic DNA fragments and protein at very low
constant high level plateau. Therefore the typical reaction history levels. The enormous amplification power of kinetic PCR may
can be divided in four characteristic phases, Figure 1. The 1st result in even the smallest amount of DNA contamination
phase is hidden under the background fluorescence, where an interfering with the desired specific RT-PCR product. To confirm
exponential amplification is expected; 2nd phase with exponential the absence of residual DNA, either a negative control (minus-RT
amplification that can be detected and above the background; 3rd or water) should be included in each experimental setup. It may
phase with linear amplification efficiency and a steep increase of be necessary to treat the RNA sample with commercially available
fluorescence; and finally 4th phase, or plateau phase, defined as RNase-free DNase, to get rid of any DNA. However, unspecific
the attenuation in the rate of exponential product accumulation, side reactions of the DNase often result in RNA degradation.
normally seen in later cycles. However, it is always recommended to remove the DNase prior to
any RT or PCR step. Furthermore, the design of the PCR product
Figure 1 should incorporate at least one exon-exon splice junction to allow
60 a product obtained from the cDNA to be distinguished on
electrophoresis from genomic DNA contamination.
50
4th phase RNA Quantity and Integrity
40
Accurate quantification and quality assessment of the starting RNA
3rd phase
sample is particularly important for an absolute quantification
30
strategy that normalises specific mRNA expression levels against a
given calibration curve measured in molecules or concentrations/
2nd phase grams RNA. The RNA quality assessment requires accurate quanti-
20
fication of the isolated total RNA or mRNA fraction by optical
background level density at 260 nm, (OD260), and determination of the RNA quality
10
1st phase
calculated by the OD260/OD280 ratio or by the RiboGreen RNA
Quantification Kit from Molecular Probes. Furthermore, the RNA
0
0 10 20 30 40
quality can be verified by capillary electrophoresis with the
Real-time PCR cycle Bioanalyzer 2100 on a microchip lab-on-chip system from Agilent
Technologies. The recently developed RNA integrity number (RIN)
The four characteristic phases of PCR. determines the level of intact total RNA on the basis of an
electropherogram, Figure 2. The RIN value can range between
The following information addresses optimisation strategies in 1-10: RIN 1 for totally degraded RNA, and RIN 10 for a perfect
quantitative real-time RT-PCR. Special focus is laid on the pre- and intact total RNA. RIN numbers under 5 are at least partly
analytical steps, sampling techniques, RNA extraction, and reverse degraded and result in low 18S rRNA and 28S rRNA peaks in the
transcription (RT), primer usage, and post-analytical steps, especially electropherogram. In a broader sense, poor RNA quality and low
on crossing point evaluation and crossing point measuring at RIN numbers influence the qRT-PCR performance significantly and
elevated temperatures. lead to an inhibition of the PCR performance in general and to a
Tissue Sampling and RNA Extraction later crossing point. Therefore, the extracted total RNA quality
and quantity must be verified prior to qRT-PCR experiments to
The sampling and preparation of intact cellular total RNA or
end in reliable mRNA quantification results.
mRNA is critical to all gene expression analysis techniques. A
successful and reliable experiment needs high quality, DNA free,
undegraded RNA. The source of tissue sampling techniques and
the subsequent storage of the tissue material often varies
significantly between processing laboratories (e.g. biopsy
material, single cell sampling, laser micro-dissection, slaughtering
samples). Some researchers store the tissue sample in RNAlater ®

Our Innovation, Your Research — Shaping the Future of Life Science 35

 Appendix 2
Figure 2 To circumvent these high inter-assay variations in RT, target gene
unspecific primers, e.g. random-hexamer, -octamer or -decamer
primers, can be used and a cDNA pool can be synthesised.
Similarly, poly-T oligo-nucleotides (consisting solely of 16-25
Appendix 2

desoxythymidine residues) can anneal to the poly-adenylated

3-’ (poly-A) tail found on most mRNAs. cDNA pools synthesised
with unspecific primers can be split into a number of different
target-specific kinetic PCR assays. This maximises the number of
genes that can be assayed from a single cDNA pool derived from
one small RNA sample. Therefore, the gene expression results are
directly comparable between the applied assays, at least within
one and the same RT pool. In conclusion, a rank order of RT
efficiency can be shown for the applied different primers for one
specific gene: random hexamer primers > poly-dT primer > gene
specific primer (M. Pfaffl, unpublished results).

An electropherogram of total RNA is shown (178 ng/µl total RNA; RIN = 7.9; : Elevated Fluorescence Acquisition
ratio 28s/18s = 1.4). The characteristic total RNA profile represent an internal
Real-time assays using SYBR Green I binding dye can easily reveal
reference peak (22 s), a small 5S RNA peak (27 s), a dominant 18S RNA peak
(41 s) and a dominant 28S RNA peak (43 s). the presence of primer dimers, which are the product of non-
specific annealing and primer elongation events. These events
Reverse Transcription take place as soon as PCR reagents are combined in the tube.
During PCR, formation of primer dimers competes with formation
An optimal reverse transcription (RT) is essential for a reliable and
of specific PCR product, leading to reduced amplification
successful qRT-PCR assay. The RT step is the source of high variability
efficiency and a less successful specific RT PCR product. To
in a kinetic RT-PCR experiment and for each enzyme specific reaction,
distinguish primer dimers from the specific amplicons, a melting
conditions have to be optimised. Buffer salt contamination, pH,
curve analysis can be performed in all available quantification
fatty acids, alcohol, phenol and other chemical or enzyme inhibitors
software. The pure and homogeneous RT-PCR product produces
carried over from the RNA isolation process can affect the apparent
a single, sharply defined melting curve with a narrow peak. In
RT efficiency. The extracted total RNA may contain chemical or
contrast, the primer dimers melt at lower temperatures (< 80 °C)
enzymatic tissue inhibitors that result in reduced RT and PCR
and have diffuse and broader peaks. To get rid of primer dimers,
reaction efficiencies and generate unreliable quantification results.
an intensive primer optimisation is needed by testing multiple
For many quantitative applications, MMLV H minus RT is the enzyme
primer pair by cross-wise combinations. Multiples of such primer
of choice, as the cDNA synthesis rate can be up to 10-fold greater
optimisation strategies have been developed.
than that of AMV. In numbers, the MMLV H minus RT has the ability
to reverse transcribe between 50 and 80% of the total RNA and The easiest and most effective way to get rid of any dimer
the AMV only around 5-20%. Newly available thermo-stable RNAse H structures, at least during the quantification procedure, is to add
minus RT maintains its activity up to 70 °C, thus permitting increased an additional 4th segment to the classical three segmented PCR
specificity and efficiency of first primer annealing. procedure, Figure 3: 1st segment with denaturation at 95 °C;
2nd segment with primer annealing at 60 °C; 3rd segment with
Another source of variability is the choice of priming method
elongation at 72 °C; 4th segment with fluorescence acquisition at
used to initiate cDNA synthesis, which can be either target gene-
elevated temperatures (herein 85 °C). The fluorescence acquisition
specific or non-specific. Target gene specific primers work well in
in the 4th segment eliminates the non-specific fluorescence signals
conjunction with elevated RT reaction temperatures to eliminate
derived by primer dimers or unspecific minor products and ensures
spurious transcripts. The same reverse primer is used for the
accurate quantification of the desired qRT-PCR product. High
subsequent PCR assay in conjunction with the corresponding
temperature quantification keeps the background fluorescence
gene-specific sense primer (forward primer); however, the use of
and the ‘no-template control’ fluorescence under 10% of maximal
gene-specific primers necessitates a separate RT reaction for each
fluorescence at plateau and ensures an optimal dynamic fluores-
gene of interest. It cannot be assumed that different reactions
cence range.
have the same cDNA synthesis efficiencies. The result can be high
variability during multiple RT reactions.

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Figure 3 acceleration of fluorescence. The amplification reaction and the

100
kinetic fluorescence history over various cycles is obviously not a
50
72 °C smooth and easy function. The mathematical algorithm on which
the “second derivative maximum method” is unpublished, but it

Appendix 2
is possible to fit sigmoidal and polynomial curve models, with
20
fluorescence (10log)

3-step vs. 4-step

high significance, p<0.001, and coefficient of correlation, r>0.99.
10

real-time qPCR 5 negative control

10^9 molecules
10^8 molecules
10^7 molecules
This increase in the rate of fluorescence increase, or better called
2
10^6 molecules
10^5 molecules
the acceleration of the fluorescence signal, slows down at the
1
0 5 10 15 20
cycles
25 30
10^4 molecules
10^3 molecules beginning of the 3rd linear phase. Therefore the cycle where the
10^2 molecules
100
85 °C 10^1 molecules 2nd derivative is at its maximum is always between 2nd exponential
50
and 3rd linear phase. Here, above the background level within the
85 °C real exponential phase and very close to the background line, the
fluorescence (10log)

20

10 optimal CP should be placed.

72 °C 5

1
0 5 10 15 20 25 30 35 40 45 50
cycles

Advantage of elevated real-time PCR fluorescence acquisition temperature.

Crossing Point Data Evaluation

The amount of amplified target is directly proportional to the
input amount of target only during the exponential phase of PCR
amplification. Hence the key factor in the quantitative ability of
kinetic RT-PCR is that it measures the product of the target gene
within that phase. The data evaluation, or crossing points (CP) or
threshold cycle (Ct) determination is very critical to the users. For
CP determination, various fluorescence acquisition methodologies
are possible. The “fit point method” (e.g. in LightCycler software)
and the “threshold cycle method” (used in most quantification
software) measure the CP at a constant fluorescence level. These
constant threshold methods assume that all samples have the
identical synthesised DNA concentration at the point where the
fluorescence signal significantly increases over the background
fluorescence, 2nd to 3rd phase in Figure 1. Measuring the level
of background fluorescence can be a challenge in real-time PCR
reactions with significant background fluorescence variations,
caused by drift-ups and drift-downs over the course of the
reaction. Averaging over a drifting background will give an
overestimation of variance and thus increase the threshold level.
The threshold level can be calculated by fitting the intersecting
line upon the ten-times value of ground fluorescence standard
deviation, e.g., Applied Biosystems software. This acquisition
mode can be easily automated and is very robust. In the “fit point
method” the user has to discard the uninformative background
points, exclude the plateau values by entering the number of
log-linear points, and then fit a log-line to the linear portion of
the amplification curves. These log lines are extrapolated back to
a common threshold line and the intersection of the two lines
provides the CP value. The strength of this method is that it is
extremely robust. The weakness is that it is not easily automated
and so requires a lot of user input. The problems of defining a
constant background for all samples within one run, sample-to-
sample differences in variance, and absolute fluorescence values
led to the development of new acquisition modus according
to mathematical algorithms. In the LightCycler software the
“second derivative maximum method” is performed in which
CP is automatically identified and measured at the maximum

Our Innovation, Your Research — Shaping the Future of Life Science 37

 References
1. Higuchi, R., et al., Kinetic PCR analysis: Real-time monitoring of DNA ampli- 16. Steuerwald, N., et al., Analysis of gene expression in single oocytes and
fication reactions. Biotechnology, 11, 1026 (1993). embryos by real-time rapid cycle fluorescence monitored RT-PCR. Mol. Hum.
2. Pals, G., et al., A rapid and sensitive approach to mutation detection using Reprod., 5, 1034-1039 (1999).
real-time polymerase chain reaction and melting curve analyses, using 17. Vet, J.A., et al., Multiplex detection of four pathogenic retroviruses using
BRCA1 as an example. Mol Diagn., 4, 241-246 (1999). Molecular Beacons. Proc. Natl. Acad. Sci. USA, 96, 6394-6399 (1999).
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3. Selvin, P.R., Fluorescence resonance energy transfer. Meth. Enzymol., 246, 18. Solinas, A., et al., Duplex Scorpion primers in SNP analysis and FRET applica-
300-334 (1995). tions. Nucleic Acids Res., 29, E96 (2001).
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the LightCycler. J. Biochem. Biophys. Methods, 47, 121-129 (2001). cons and fluorescence. Nat. Biotechnol., 17, 804-807 (1999).
5. Bonnet, G., et al., Thermodynamic basis of the enhanced specificity of 20. Thelwell, N., et al., Mode of action and application of Scorpion primers to
structured DNA probes. Proc. Natl. Acad. Sci. USA, 96, 6171-6176 (1999). mutation detection. Nucleic Acids Res., 28, 3752-3761 (2000).
6. Broude, N.E., Stem-loop oligonucleotides: A robust tool for molecular biol- 21. Hart, K.W., et al., Novel method for detection, typing, and quantification
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