qPCR

guide
 3

preface

Real-Time quantitative PCR, or qPCR in short, is heralded as the gold

standard for accurate, sensitive and fast quantification of nucleic acid
sequences. Indeed it is a wonderful technology, simple to perform and
the risk for cross contamination is extremely low, as reaction tubes do not
need to be opened. However, the simplicity of generating data is also the
Achilles heel of qPCR. Nowadays, high quality reagents are available, and
dedicated instruments do a perfect job. Hence, anyone that masters the
skill of pipetting can generate beautiful sigmoidal amplification curves.
What many investigators unfortunately fail to appreciate is quality control,
which is essential throughout the entire qPCR workflow (from living cells, over extraction of
nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription
and PCR amplification, and finally data-analysis) in order to draw biologically meaningful
conclusions. It is now well over 10 years after the conception of qPCR and there is still no
consensus on good laboratory practice in general, and the level of quality control, experiment
design and the data-analysis strategy in particular. As such, the life science literature is flawed
with studies that are meaningless, inconclusive, or in the worst case erroneous (resulting in
retraction of some papers). Fortunately, standards for data exchange and reporting guidelines
are finding their way to the scientific community (see for example efforts made by the RDML
consortium, http://www.rdml.org).

When inspecting the scientific literature relating to qPCR, it is clear that the number of publications
over the years follows the same exponential rate as the PCR reaction itself. Currently, there is
no plateau phase in sight; qPCR remains a growing market, especially in clinical diagnostics.
In the research field, gene expression analysis is by far the most popular application, with
double stranded DNA specific binding dyes and hydrolysis probes as the dominating
detection chemistries (source: the Real-Time PCR Primer and Probe database, RTPrimerDB,
http://medgen.ugent.be/rtprimerdb/).

Coming from relatively low throughput qPCR systems a decade ago, 384-well instruments have
now become more and more mainstream. “New kids” are now also on the block, implementing
nanofluidic technologies, significantly increasing throughput and decreasing reaction volumes
(down to nanoliter range), without the need for advanced liquid handling instruments. Following
this promising path of ever increasing throughput, we might foresee a future in which we will
simply PCR profile entire signalling networks or pathways, or even determine the expression
level of almost every gene in an organism within only a few hours, with unprecedented sensitivity,
accuracy and at low cost. Surely, it’s a long road with many hidden obstacles, but witnessing
the tremendous progress the science community have made in the last decade, the envisioned
whole genome PCR profiling comes within reach.

Jo Vandesompele
Professor functional genomics and applied bio-informatics, Ghent University
Co-founder and CEO, Biogazelle, the Real-Time PCR data analysis company

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 qPCR guide

Table of contents

Introduction 6
Preliminary steps 7
Template preparation: DNA or RNA 7
One-step or two-step reaction? 8
Which type of primers for the Reverse Transcription? 9
Qualitative vs quantitative PCR technologies 10
What are qualitative PCR & quantitative PCR? 10
Quantitative PCR 11
Qualitative PCR 11
Detection methods 12
SYBR® Green I 12
High Resolution Melting dyes (HRM dyes) 13
TaqMan® probes = Double-Dyes probes 13
LNA Double-Dye probes
®
15
Molecular Beacon probes 16
Scorpions primers
®
18
Hybridization probes (also called FRET probes) 19
TaqMan MGB probes
® ®
20
MGB Eclipse® probes 20
Other technologies 21
When should I multiplex? 21
How do you get started in qPCR? 22
Thermocycler 22
qPCR reagents : formats and components? 24
Core kits or MasterMixes? 24
Role of core kits and MasterMixes components 24
Probe and primer quality 26
Manual coupling 26
Automatic coupling 26
Purification level 26

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Guidelines for primers and probes design 27

General rules 27
Design guidelines for SYBR® Green I assays 28
Design guidelines for Double-Dye Probe assays 29
Quenching principle 31
Fluorophore and quencher: definitions 31
Different ways of quenching 31
FRET quenching 32
Optimal flurophore quencher combination 32
Dye-thermocyclers compatibility table 34
Double-Dye Oligonucleotides in single-color detection 34
Double-Dye Oligonucleotides for multiplexing detection 34
Example of a classical assay 36
Some tips and tricks before starting 36
Classical protocol and thermal profile 37
Useful definitions 38
Normalization and quantification methods 39
Absolute quantification 41
Relative quantification 41
Evaluation of your qPCR assay 43
Controls 43
Negative controls 43
Positive controls 44
Commonly used control kits 45
Singleplex or multiplex assay for the controls? 45
Role of the standard curve and evaluation of its parameters 46
PCR efficiency 46
R square (R²) 47
Sensitivity 47
Reproductibility 47
Specificity of a SYBR Green I assay
®
47
Optimization tips 49
In general 49
For probe multiplexing assay/qualitative qPCR 50
Troubleshooting guide 51
Frequently Asked Questions 53
Eurogentec products 55
References 64

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 qPCR guide

This guide is dedicated for both beginners and experts in qPCR. It explains both the basics of
qPCR and gives useful tips for troubleshooting. Real-Time qPCR is a technique that involves a
high number of parameters, all of them having to be optimized to get the highest quality assay,
and all of which are specific to one assay only. There is consequently no “magic formula”.
However, this booklet can provide tools to help you, from your first contact with qPCR to the
optimization steps. If you are an expert, just go straight to the last pages of this booklet. We
hope that by working through the pages, we will help you enjoy this technique and reach the
best results for your research!

Introduction

The Polymerase Chain Reaction (PCR) has been invented in 1983 by Kary
Mullis (Nobel Price in 1993), (Mullis K. et Fobona F., 1987). Three years after
its invention, there was an incredible expansion of its use thanks to the
commercialization of the Taq polymerase, a polymerase that resists high temperatures.
In 1991, the first hydrolysis probe was used in combination with the technique.
In 1992, the technique was again improved by the use of Ethidium Bromide (EtBr),
thanks to the fluorescence that results from the binding to duplex DNA. The kinetics
of fluorescence accumulation during thermocycling was directly related to the starting
number of DNA copies. This was the starting point of Real-Time qPCR. Today, 25
years after Mullis’s discovery, both PCR and qPCR are widely used technologies.
The principle, and aim, of the PCR technology is to specifically increase a target from an
undetectable amount of starting material. In classical PCR, at the end of the amplification,
the product can be run on a gel for detection of this specific product. In Real-Time PCR, this
step can be avoided since the technology combines the DNA amplification with the immediate
detection of the products in a single tube. The homogeneous format is highly beneficial as it
removes the significant contamination risk caused by opening tubes for post-PCR manipulation.
It is also less time consuming than gel based analysis and can give a quantitative result (Kubista
M. et al., 2006).

Current detection methods are based on changes in fluorescence, which are proportional to the
increase of target. Fluorescence is monitored during each PCR cycle providing an amplification
plot, allowing the user to follow the reaction in real time. (Figure 1).

Delta Rn VS Cycle
7000

6000

5000

4000
Delta Rn

3000

2000

1000

-1000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Cycle Number

Figure 1. Amplification plot of 10 x serial diluted cDNA, using SYBR® Green detection method, on an ABI 7500
thermocycler.

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Preliminary steps

Variability in qPCR is often related to steps upstream to the qPCR step itself. Sample extraction,
quantity of sample, or efficiency of the reverse transcription (RT) are some of the many
parameters that can influence the results of your qPCR assay.

That’s why it is important to also consider the quality of these steps prior to performing your
qPCR assay (Fleige S. and Pfaffl MW, 2006).

Template preparation: DNA or RNA

DNA and RNA can be extracted with Trizol. This includes many steps and a careful wash of
the sample, hence we recommend the use of an appropriate commercially available kit. Many
column-based kits, which will contain all the required reagents for the full extraction / purification
procedure are available.

These kits will also outline general guidelines, such as the storage conditions and shelf life of
the extracted RNA or DNA. However these guidelines may vary between kits due to the different
composition of buffers. It is also recommended that the buffers supplied with each kit are used
according to the manufacturer protocol.

The final product should be cleaned and free from any residual buffers such as EDTA or buffers
containing solvents. Otherwise, these residual products may inhibit the action of the Taq
polymerase for the PCR step, or could modify the salt concentration of the buffer. This is usually
not a problem when using spin column kits instead of manual extraction techniques.

It is worth bearing in mind, that this first step is nearly the most important of your RT-qPCR assay,
as the quality of the extraction will influence the quality of your detection and quantification.
It’s mostly important to ensure the reproducibility of your extraction steps, if you wish to compare
biological samples.

The main problems that can occur, are extraction of inhibitors together with the nucleic acids
or degradation of the sample. Inhibitors are mainly found in blood sample or environmental
samples. For example, humic acid can be a strong inhibitor in samples extracted from soil.
As a general rule, it is always better to extract DNA/RNA from fresh sample and to store it
at -80 °C.

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 qPCR guide

One-step or two-step reaction?

If your starting material is total RNA, you have to perform the Reverse Transcription (RT) step
before your qPCR assay. This can be done in One-Step or Two-Steps. Both methods have pros
and cons you might consider to choose the best one for your application.
In both cases, the RT reaction should be set up in a clean environment to avoid contamination.
We also recommend working with RNase free plastics. The tubes containing the reaction should
be maintained on ice during the set up of the reaction. This will prevent an early reaction,
generated before the incubation period.

The RT step lasts 30 minutes around 50 °C. However, if random primers or oligo d(T) are used
(two-step reaction), an initial 10 min-step at 25 °C is necessary to maximize the annealing
efficiency, as the short oligo d(T) have a lower Tm. The next paragraph explains what kind of
primers can be used under which circumstancies.

The cDNA generated should be stored at -80 °C. The use of a separate RT step is recommended
when the reaction is performed with a limiting amount of starting material.

One-Step qRT-PCR Two-step qRT-PCR

Description RT and qPCR reactions performed in RT and qPCR reactions performed in separate tubes
the same tube

Optimized working buffer for both

the RT and PCR enzymes

Pros Save pipetting steps More efficient because random primers and oligo d(T)
can be used
No contamination between RT and Possibility to stock cDNA to quantify several targets
qPCR steps

Lower background in SYBR® assays More flexible (separate optimization possible for the
two reactions)
Best option for High-Throughput
Screening (less time consuming
than 2-step reactions)

Cons No possibility to use UNG carry-over RNase inhibitors that can influence the PCR reaction
prevention after the RT
Usually less sensitve than a Two- Higher background when performing a SYBR® assay
Steps qRT-PCR assay

Table 1: Pros and cons of One-Step qRT-PCR versus Two-Step qRT-PCR

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Which type of primers for the Reverse Transcription?

When performing a RT, it is possible to use three different types of primers:

• short random primers, which bind anywhere in the genome and allow the reverse
transcriptase to fill up the gaps, lead to higher yields
• oligo d(T) bind to the poly-A tail of the RNA and then only transcribe RNA. This
will avoid contamination by genomic DNA. As the poly-A tail is located at the
extremity of the gene, it will also lead to more full transcripts
• specific primers, which bind to the gene of interest

The combination of oligo d(T) primers and random primers will give the highest yields and
the longest transcripts, whereas specific primers transcribe only specific RNA but reduce
the yield.

With a One-Step qRT-PCR reaction, using specific primers is the only option, as it should be
avoided that oligo d(T) primers and random nonamers participate in the PCR step, giving many
aspecific products. The RT step is performed at 40-50 °C and the specific PCR primers are
designed for the PCR step at 60 °C, leading to partial annealing of the primers during the RT
step. In a Two-Steps qRT-PCR kit, oligo d(T) primers or/and random nonamers are used in the
RT step, and specific primers in the PCR step, leading to specific cDNA.

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 qPCR guide

Qualitative vs. Quantitative PCR

What are qualitative PCR & quantitative PCR?

Firstly it is important to review the principles of PCR, before making the distinction between
qualitative and quantitative PCR.

The action of the Taq polymerase allows extension of short single-stranded synthetic
oligonucleotides (i.e. primers) during repeated cycles of heat de-naturation, primer annealing
and primer extension.

The primers are designed to specifically bind the extremities of the DNA fragment to be
amplified. The Taq polymerase uses the target DNA added to the reaction as a template for
primer extension. At each cycle, more DNA is synthesized, creating more template DNA. The
reaction proceeds in an exponential manner, doubling the amount of target during each cycle,
until one of the reagents becomes limiting and the reaction reaches a plateau.

Figure 2. The Polymerase Chain Reaction. (K. Mullis and F. Fabona, 1987). The red and pink lines represent the
primers that the polymerase extends from. The blue and black lines represent the single-stranded DNA template
produced from de-naturation of the double-stranded DNA template.

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Quantitative PCR
In quantitative qPCR, a specific or non-specific detection chemistry allows the quantification of
the amplified product. The amount detected at a certain point of the run is directly related to the
initial amount of target in the sample.

The most common applications of quantitative PCR are gene expression analysis, pathogen
detection/quantification and microRNA quantification (Schmittgen TD et al.,2008).

For example, effects of different treatments, on the level of mRNA transcription can be measured.
Quantitative PCR software uses the exponential phase of PCR for quantification. PCR is initially
an exponential process but eventually reaches a plateau phase, when one of the reagents
becomes limited. Reactions can plateau at different levels even if they have the same starting
concentration of target. During the exponential phase, the amount of target is assumed to be
doubling every cycle and no bias is expected due to limiting reagents. Analysis takes the Ct
(cycle number) value, at the point when the signal is detected above the background and the
amplification is in exponential phase. The more abundant the template sample, the quicker this
point is reached, thus giving earlier Ct values. Differences in Ct then have to be correlated to
some other quantitative values to make them meaningful (cf. p. 39).

Qualitative PCR
In qualitative qPCR, the goal is to detect the presence or absence of a certain sequence. It could
be for virus sub-typing and bacterial species identification for example. It can also be used for
allelic discrimination between wild type and mutant, between different SNPs (Single Nucleotide
Polymorphisms) or between different splicing forms. In this case, different fluorophores can
be used for the two alleles, and the ratio of the fluorophores signals correlates to the related
amount of one form compared to the other one.

Specific detection methods such as Double-Dye probe systems are more often used for these
applications, and probes can be used to detect single base mutations or small deletions.

Recently, a new technique called HRM (High Resolution Melting) emerged, allowing gene
scanning and SNP genotyping without using probes (cf. p. 13).

SNP “Y” Only

Allele Y Fluorescent Signal

Both “X” and “Y” SNPs

SNP “X” Only

Allele X Fluorescent Signal

Figure 3. Allelic discrimination using two Double-Dyes labelled with different fluorophore (ncvs.org).

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 qPCR guide

Detection methods
In order to detect and measure the amount of target in the sample, a measurable signal has to
be generated, which is proportional to the amount of amplified product. All current detection
systems use fluorescent technologies.

Some of them are non-specific techniques, and consequently only allow the detection of one
target at a time. Alternatively, specific detection chemistries can distinguish between non-
specific amplification and target amplification. These specific techniques can be used to
multiplex the assay, i.e. detecting several different targets in the same assay.

SYBR® Green I
SYBR® Green I is the most commonly used dye for non-specific detection. It is a
double-stranded DNA intercalating dye, that fluoresces once bound to the DNA. A pair of specific
primers is required to amplify the target with this chemistry. The amount of dye incorporated
is proportional to the amount of generated target. The dye emits at 520 nm and fluorescence
emitted can be detected and related to the amount of target.

The inconvenience of this technique is that the SYBR® Green I will bind to any amplified dsDNA.
Consequently, primer dimers or unspecific products introduce a bias in the quantification.
However, it is still possible to check for the specificity of the system by running a meltcurve at
the end of the PCR run (cf. p. 47). The principle is that every product has a different dissociation
temperature, depending of the size and base contents, so it is still possible to check the number
of products amplified. A valid SYBR® assay - primer pair - should produce a unique, well defined
peak on the meltcurve.

For these reasons, SYBR® Green I is rarely used for qualitative PCR. However, SYBR® Green I
is often used as the first step to optimize a specific detection system assay, to check the
specificity of the primers and validate the design.

SYBR® Green I advantages

• Low cost assay

• Easy design and set up

SYBR® Green I disadvantages

• Non specific system

• Not adapted to multiplex
• Non suitable for qualitative qPCR

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High Resolution Melting dyes (HRM dyes)

High Resolution Meltcurve analysis is a newly emerging technology, which characterizes nucleic
acid samples based on their dissociation behaviour. It combines the principle of intercalating
dyes, meltcurve analyses and the application of specific statistical analyses.

HRM uses the fundamental property of the separation of the two strands of DNA with heat
(melting), and the monitoring of this melting with a fluorescent dye. On the contrary of SYBR
Green, HRM dyes do not inhibit PCR at high concentration. The dye can consequently saturate
the amplified target dsDNA and fluoresces. Melting temperature of a dsDNA target depends on
GC content, length, and sequence. Due to the high sensitivity of HRM dyes, even a single base
change will induce differences in the melting curve, and consequently in fluorescence (Erali M.
et al., 2008).

Main applications of HRM include gene scanning (search for the presence of unknown
variations in PCR amplicons), SNP genotyping, DNA methylation analysis, DNA mapping, DNA
fingerprinting.
This emerging method is less expensive and as precise than probe-based methods.

Only a few thermocyclers on the market currently allow the use of this technology, among them
the Roche LightCycler®480, the Corbett Life Science Rotor-Gene™ 6000, and the ABI Prism®
7500. The main HRM dyes available are EvaGreen, LCGreen®, SYTO® 9 and BEBO.

TaqMan® probes = Double-Dye probes

TaqMan® probes, also called Double-Dye Oligonucleotides, Double-Dye Probes, or Dual-
Labelled probes, are the most widely used type of probes and are often the method of choice
for scientists who have just started using Real-Time PCR. They were developed by Roche
(Basel, Switzerland) and ABI (Foster City, USA) from an assay that originally used a radio-
labelled probe (Holland et al. 1991), which consisted of a single-stranded probe sequence that
was complementary to one of the strands of the amplicon.

A fluorophore is attached to the 5’ end of the probe and a quencher to the 3’ end. The fluorophore
is excited by the machine and passes its energy, via FRET (Fluorescence Resonance Energy
Transfer) to the quencher. Traditionally the FRET pair has been FAM as the fluorophore and
TAMRA as the quencher. In a well designed probe (cf. Taqman® design rules on p. 30), FAM
does not fluoresce as it passes its energy onto TAMRA. As TAMRA fluorescence is detected
at a different wavelength to FAM, the background level of FAM is low. The probe binds to the
amplicon during each annealing step of the PCR. When the Taq polymerase extends from the
primer which is bound to the amplicon, it displaces the 5’ end of the probe, which is then
degraded by the 5’-3’ exonuclease activity of the Taq polymerase. Cleavage continues until the
remaining probe melts off the amplicon (cf. fig. 4).

This process releases the fluorophore and quencher into solution, spatially separating them
(compared to when they were held together by the probe). This leads to an irreversible increase
in fluorescence from the FAM and a decrease in the TAMRA.

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 qPCR guide

Figure 4. TaqMan® probes mode of action

TaqMan® probes can be used for both quantification and mutation detection, and most designs
appear to work well. They are convenient either for allelic discrimination or expression profiling
and are usually easy to design, easy to use in a standard protocol and need minimal optimization.
For mutation detection, the probe is designed to hybridize over the mutation site and can be
made specific enough to detect single base differences.

To obtain robust allelic data, it is vital that a different probe is used for each different mutation,
otherwise negative results with one probe, (caused by failed PCR reactions), can be scored as
an absence of a particular allele. Ideally these probes would be multiplexed to make the assays
faster and cheaper.

Double-Dye probes advantages

• Widely used, several modifications possible

• Specific system
• Multiplex capabilities

Double-Dye probes disadvantages

• More expensive than SYBR® Green I, but the lowest cost specific
system available

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LNA® Double-Dye probes

LNA® (Locked Nucleic Acid) was developed by Exiqon® (Vedbaek, Denmark). LNA® changes
the conformation of the helix and increases the stability of the duplex. The integration of LNA®
bases into Double-Dye Oligonucleotide probes, opens up great opportunities to improve
techniques requiring high affinity probes as specific as possible, like SNP detection, expression
profiling and in situ hybridization.

LNA® is a bicyclic RNA analogue, in which the ribose moiety in the sugar-phosphate backbone
is structurally constrained by a methylene bridge between the 2’-oxygen and the 4’-carbon
atoms.

O Base O
Base
O O
O

O O O
O P O'
O P O'

LNA
Figure 5. LNA® backbone structure

The integration of LNA® bases into probes changes the conformation of the double helix from
the B to A type (Ivanova A. et al., 2007).

Figure 6. B helix and A helix containing LNA® bases

LNA® conformation allows a much better stacking and therefore a higher stability. By increasing
the stability of the duplex, the integration of LNA® monomers into the oligonucleotide
sequence allows an increase of the melting Temperature (Tm) of the duplex. It is therefore
possible to reduce the size of the probe, which increases the specificity of the probe and helps
designing it (Karkare S. et al., 2006).

LNA® advantages

• Increases thermal stability towards complementary DNA and RNA

• Allows use of shorter probes
• High specificity and reproducibility

• Simplifies multiplex assays by adjusting Tm values of primers and probes

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 qPCR guide

Molecular Beacon probes

Molecular Beacons are probes that contain a stem-loop structure, with a fluorophore and a
quencher at their 5’ and 3’ ends, respectively. The stem is usually 6 bases long, should mainly
consist of C’s and G’s, and holds the probe in the hairpin configuration (Li Y. et al., 2008).
The ‘stem’ sequence keeps the fluorophore and the quencher in close vicinity, but only in the
absence of a sequence complementary to the ‘loop’ sequence. As long as the fluorophore
and the quencher are in close proximity, the quencher absorbs any photons emitted by the
fluorophore. This phenomenon is called collisional (or proximal) quenching. In the presence of
a complementary sequence, the Beacon unfolds and hybridizes to the target, the fluorophore
is then displaced from the quencher, so that it can no longer absorb the photons emitted by
the fluorophore, and the probe starts to fluoresce. The amount of signal is proportional to the
amount of target sequence, and is measured in real time to allow quantification of the amount
of target sequence (Takacs T. et al., 2008).

The increase in fluorescence that occurs is reversible, (unlike TaqMan® probes), as there is no
cleavage of the probe, that can close back into the hairpin structure at low temperature.

The stem structure adds specificity to this type of probe, because the hybrid formed between
the probe and target has to be stronger than the intramolecular stem association. Good design
of Molecular Beacons can give good results, however the signal can be poor, as no physical
separation of fluorophore from quencher occurs.

20 000

Beacon-target hybrid
15 000
Fluorescence

10 000

5 000

Beacon without target

0

15 35 55 75

Temperature (°C)

Figure 7. Melt curve of a Molecular Beacon with and without a synthetic complement as a target. Annotations show
the configuration of the beacon and target at different temperatures.

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Two improved forms of Molecular Beacons exist:

• Wavelength-Shifting Molecular Beacons

Wavelength-Shifting Molecular Beacons are brighter than standard Molecular Beacons due
to an enhanced fluorescence intensity of the emitter fluorophore. These probes contain a
harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic
light source, an emitter fluorophore of the desired emission color, and a non-fluorescent
(dark) quencher. In the absence of complementary nucleic acid targets, the probes are non-
fluorescent, whereas in the presence of targets, they fluoresce, not in the emission range
of the harvester fluorophore, that absorbs the light, but rather in the emission range of the
emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed
energy from the harvester fluorophore to the emitter fluorophore by FRET, which only takes
place in probes that are bound to the targets. Wavelength-Shifting Molecular Beacons are
substantially brighter than conventional Molecular Beacons that cannot efficiently absorb
energy from the available monochromatic light source (Tyagi S. et al., 2000).

• 2’ O-Methyl RNA Molecular Beacons

To detect the various RNA classes in living cells, several approaches have been developed.
One of these is based on the use of 2’-O-Methyl RNA Molecular Beacon probes for the
detection of small nuclear RNAs. 2’-O-methyl RNA probes are considered to perform
better than DNA oligonucleotides because they are not only nuclease resistant, but also
possess a higher affinity, increased specificity, faster hybridization kinetics, and a superior
ability to bind to structured targets compared to DNA oligonucleotides. Recently Molecular
Beacons were introduced for RNA detection in living cells. The rationale for using Molecular
Beacons to detect RNAs in living cells was to improve signal-to-noise ratios by eliminating
fluorescence signals derived from non-hybridized probe sequences.
It appears that linear 2’-O-Methyl RNA probes are usually more suitable for specific
detection of these RNAs, representing different classes of RNA, in the nuclei of living cells.
Molecular Beacons result in images with improved signal to noise ratios, thereby leading to
better detection sensitivity.

Molecular Beacons

• Reversible phenomenon (melting curve possible)

• Increased discriminatory competence due to competition intra-inter target

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 qPCR guide

Scorpions® primers
Scorpions® primers are suitable for both quantitative Real-Time PCR and genotyping/end-point
analysis of specific DNA targets.
They are PCR primers with a “stem-loop” tail consisting of a specific probe sequence, a
fluorophore and a quencher. The “stem-loop” tail is separated from the PCR primer sequence
by a “PCR blocker”, a chemical modification that prevents the Taq polymerase from copying the
stem loop sequence of the Scorpions® primer. Such read-through would lead to non-specific
opening of the loop, causing a non-specific fluorescent signal.

The hairpin loop is linked to the 5’ end of a primer via a PCR blocker. After extension of the primer
during PCR amplification, the specific probe sequence is able to bind to its complement within
the same strand of DNA. This hybridization event opens the hairpin loop so that fluorescence is
no longer quenched and an increase in signal is observed.

HXdge^dchœeg^bZg

* ( * ( ;
; F
iVg\Zi ]ZViidYZcVijgZ
* ( F

( *
eg^bZg

XddaVcYVccZVaeg^bZgh

* ( * ( * ( * (
; F ZmiZch^dce]VhZ ; F
( * ( *

]ZViidYZcVijgZ

XddaVcYYZ[ZXi[ajdgZhXZcXZ

F ;
(
F

E8G7adX`Zg

Figure 8. Scorpions® unimolecular mode of action. Progression through a PCR reaction.

Unimolecular probing is kinetically favorable and highly efficient. Covalent attachment of the
probe to the target amplicon ensures that each probe has a target in the near vicinity. Enzymatic
cleavage is not required, thereby reducing the time needed for signaling compared to TaqMan®
probes, which must bind and be cleaved before an increase in fluorescence is observed.

Scorpions® primers have successfully been used for mutation detection and quantification
(Solinas A. et al., 2001), having the specificity and melt curve analysis of the Molecular Beacons,
with additional speed and efficiency. However, constraints associated with Scorpions® design is
probably the reason why this type of probe is not as successful as it should be.

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There are three types of Scorpions® primers :

• Standard Scorpions®, which consist of a bi-labelled probe with a fluorescent dye

at the 5’ end and an internal non-fluorescent quencher.
• FRET Scorpions®, for use on a LightCycler® system. As the capillary system will
only excite at 470 nm (FAM absorption wavelength) it is necessary to incorporate
a FAM within the stem. A ROX is placed at the 5’end of the Scorpions® primer,
FAM is excited and passes its energy onto the ROX.
• Duplex Scorpions® have also been developed to give much better signal intensity
than the normal Scorpions® format. In Standard Scorpions® the quencher and
fluorophore remain within the same strand of DNA and some quenching can occur
even in the open form. In the Duplex Scorpions® the quencher is on a different
oligonucleotide and physical separation between the quencher and fluorophore is
greatly increased, reducing the quenching when the probe is bound to the target.

Advantages of Scorpions® primers vs Taqman® probes

• Stronger signal and lower background

• Improved SNP applications
• Compatible with any dye, convenient for extended multiplexing assays
• Primer and probe on one molecule, kinetically favorable, fast cycling
conditions

Hybridization probes (also called FRET probes)

Roche has developed hybridization probes (Caplin et al. 1999) for use with their LightCycler®.
Two probes are designed to bind adjacent to one another on the amplicon. One has a 3’ label
of FAM, whilst the other has a 5’ LC dye, LC red 640 or 705.
When the probes are not bound to the target sequence, the fluorescent signal from the reporter
dye is not detected. However, when the probes hybridize to the target sequence during the
PCR annealing step, the close proximity of the two fluorophores allows energy transfer from the
donor to the acceptor dye, resulting in a fluorescent signal that is detected.

Figure 9. FRET probe principle

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 qPCR guide

TaqMan® MGB® probes

TaqMan® MGB® probes have been developed by Epoch Biosciences (Bothell, USA) and Applied
Biosystems (Foster City, USA). They bind to the minor groove of the DNA helix with strong
specificity and affinity. When the TaqMan® MGB® probe is complemented with DNA, it forms
a very stable duplex with DNA. The probe carries the MGB® moiety at the 3’ end. The MGB
strongly increases the probe Tm , allowing shorter, hence more specific designs.
The probe performs particularly well with A / T rich regions, and is very successful for SNP
detection (Walburger et al., 2001). It can also be a good alternative when trying to design a
probe which should be located in the splice junction (for which conventional probes are
hard to design). Smaller probes can be designed with Tm as 65-67 °C, which gives a better
discrimination (the probe is more specific for single mismatch). A good alternative to MGB
probes are LNA® probes where the increase in Tm induced by the addition of LNA® bases is
specific, contrary to the MGB moeity (cf. p. 15).

During the primer extension step, the hybridized probe is cleaved by the 5’ exonuclease activity
of Taq polymerase and an increase in fluorescence is seen. Fluorescence of the cleaved probe
during PCR is monitored in Real-Time by the thermocycler.

Figure 10. TaqMan® MGB® probe principle

MGB Eclipse® probes

MGB Eclipse® probes also known as QuantiProbes, have originally been developed by Epoch
Biosciences (Bothell, USA). MGB Eclipse® probes carry a minor groove binder moiety that
allows the use of short probes for very high specificity. These are short linear probes that have
a minor groove binder and a quencher on the 5’ end and a fluorophore on the 3’end. This is the
opposite orientation to TaqMan® MGB® probes and it is thought that the minor groove binder
prevents the exonuclease activity of the Taq polymerase from cleaving the probe. The quencher
is a Non Fluorescent Quencher also known as Eclipse Dark Quencher. Quenching occurs when
the random coiling of the probe in the free form brings the quencher and the fluorophore close
to another. The probe is straightened out when bound to its target and quenching is decreased,
leading to an increase in fluorescent signal.

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 21

Other technologies
The technologies that have been discussed above are the most widely used today, but numerous
other technologies have occurred in publications, or are available on the market, such as:
Resonsense probes, Light-up probes, HyBeacon® probes, LUX primers, Yin-yang probes, or
Amplifluor®. You can contact us for more information on any of them.

When should I multiplex?

We have previously seen that some chemistries are more adapted to multiplexing than others.
Theoretically, multiplex assay can be performed also with a non-specific system such as SYBR®
Green I assay. The most common use of multiplex reaction is for qualitative PCR assay. In a
quantitative assay, it mainly allows to include a control gene in the same well as the target
(cf. p. 39, Normalization and Quantification methods).

Multiplexing is sometimes a necessity (SNP detection), but multiplex assays can also be set up
for practical reasons, to save time and reagents (Ishii T. et al., 2007).

When is it worth multiplexing? When is it better to singleplex?

• For SNP/mutation analysis: for bi-allelic discrimination, one specific probe is

designed for each allele. Both of them have a 5’ end fluorophore and a quencher
at the 3’ end and duplex assay is necessary.
• When targeting two genes with equal expression levels with PCR reactions of
similar amplification efficiency, multiplex Real-Time PCR can be performed
without any doubt.
• When analyzing two genes with different expression levels we recommend
separating amplification of targets.
• If you quantify lots of different targets on few samples, it is not necessary to multiplex
as you won’t save time. Optimizing a multiplex reaction is time-consuming, so it
is worth doing it if you plan to work on a large cohort of samples, and always on
the same targets.
• If you have strict requirement for your design (cross linked species…), avoid
multiplexing, as you can observe annealing between probes or primers, if your
design is not optimal.
• However if you manage to have a good PCR efficiency for your singleplex assays,
if you don’t see any complementarities between your probe and primers, and if
you routinely work with the same genes, then it is worth optimizing your multiplex
reaction.

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 qPCR guide

How do you get started in qPCR?

Thermocycler
It seems logical, but you need to get a good thermocycler before you start your qPCR assay.
You will most certainly start your qPCR project with the thermocycler available in your lab, or the
lab nearby. The majority of the thermocyclers on the market now offer similar characteristics.
The table shows the main thermocyclers on the market, and can be considered as a starting
point in your decision making process.

The following characteristics should always be taken into consideration:

• Format: glass capillaries, plastics tubes, 96-well plates or 384-wells plates.

Consider this aspect depending on your requirements, and also based on the
reagent availability. A plate format is ideal if you have a high throughput requirement.
Glass capillaries can reduce the choice of reagents which you may want to use.
• Number of detection channels: if you want to multiplex, choose a thermocycler
with a large choice of dyes, in order to have the best combination for your assay.
You can find on p. 35 the table showing available dye combinations, based on the
type of thermocycler.
• Software analysis: Consider the simplicity of the software, the format to export
data, statistical analysis performed etc. In addition to qPCR analysis, you can also
perform High Resolution Meltcurve analysis with some thermocyclers.
• Time of run: the newest thermocyclers enable a very short time of run (30-45
minutes) compared to some of the older ones which take 2 hours.
• Price and flexibility of the offer. Some suppliers try to tie up the machine with their
corresponding disposable and reagents.

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 Thermocycler Supplier Format Number of channels Optical Excitation system Notes More information on:
GeneAmp® SDS 5700 Applied Biosystems 96-well plate 1 Halogen lamp Sales discontinued http://www.appliedbiosystems.com/

ABI Prism® SDS 7000 Applied Biosystems 96-well plate 4 Tungsten-halogen lamp Sales discontinued http://www.appliedbiosystems.com/

ABI Prism® SDS 7700 Applied Biosystems 96-well plate 4 Laser Sales discontinued http://www.appliedbiosystems.com/

ABI Prism® SDS 7900 HT Applied Biosystems 96-well plate or 384-well plate 4 Laser Robotic plate loading system for http://www.appliedbiosystems.com/
high throughput use
Normal or FAST block

ABI Prism® SDS 7300 Applied Biosystems 96-well plate 4 Tungsten-halogen lamp http://www.appliedbiosystems.com/

ABI Prism® SDS 7500 Applied Biosystems 96-well plate 5 Tungsten-halogen lamp Normal or FAST block http://www.appliedbiosystems.com/

StepOne® / StepOnePlus® Applied Biosystems 48-well /96-well 3/4 LEDs http://www.appliedbiosystems.com/

®
iCycler iQ BioRad 96-well plate 4 Halogen lamp Sales discontinued http://www.bio-rad.com/

My iQ® BioRad 96-well plate 1 Tungsten- halogen lamp Singleplex FAM/SYBR http://www.bio-rad.com/

iQ5® BioRad 96-well plate 5 Tungsten-halogen lamp http://www.bio-rad.com/

CFX 96® BioRad 96-well plate 5 LEDs PCR cycler converted into Real- http://www.bio-rad.com/
Time cycler

Table 2. Main characteristics of existing thermocyclers.

MiniOpticon® BioRad 48-well plate 2 LEDs http://www.bio-rad.com/

DNA Engine Opticon® 1 BioRad 96-well plate 1 LEDs Sales discontinued http://www.bio-rad.com/

DNA Engine Opticon® 2 BioRad 96-well plate 2 LEDs http://www.bio-rad.com/

®
Chromo 4 BioRad 96-well plate 4 LEDs http://www.bio-rad.com/

Mx3000P® Stratagene 96-well plate 4 Quartz tungsten-halogen lamp Customizable filters http://www.stratagene.com/

Mx3005P® Stratagene 96-well plate 5 Quartz tungsten-halogen lamp Customizable filters http://www.stratagene.com/

Mx4000® Stratagene 96-well plate 4 Quartz tungsten-halogen lamp Sales discontinued http://www.stratagene.com/

Mastercycler ®ep realplex2 Eppendorf 96-well plate 2 LEDs http://www.eppendorf.com/

®
Mastercycler ep realplex4 Eppendorf 96-well plate 4 LEDs http://www.eppendorf.com/

LightCycler® 1.5 Roche 32 glass capillaries 3 LEDs https://www.roche-applied-science.com/

LightCycler® 2 Roche 32 glass capillaries 6 LEDs https://www.roche-applied-science.com/

LightCycler 480® Roche 96-well or 384-well plate 6 Xenon lamp Optional HRM module https://www.roche-applied-science.com/

Smartcycler® Cepheid Innovation Up to 96 plastic tubes. 4 LEDs Independent block of 16 wells. http://www.cepheid.com/
Up to 6 blocks can be combined.
23

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Rotor-Gene™ 2000 / 3000 Corbett Life Science 16 / 32 or 72 plastics tubes 2/4 LEDs Sales discontinued http://www.corbettlifescience.com

Rotor-Gene™ 6000 Corbett Life Science 6 LEDs Includes HRM module http://www.corbettlifescience.com

cf. the dye-thermocycler compatibility table on p. 35

 qPCR guide

qPCR reagents : formats and components?

When choosing a reagent for your qPCR assay, consider the format and the composition, in
order to use one which is adapted to your assay, your machine, and your way of working.

Core kits or MasterMixes?

A Core kit, is a kit that contains all components in separate tubes, so you have to mix them
yourself. It gives a maximum of flexibility, as you can optimize the concentration of each
component of your assay. A qPCR Core kit contains individual tubes of reaction buffer,
dNTP/dUTP mix, MgCl2, Taq and eventually SYBR® Green.
On the contrary, a MasterMix is a ready-to-use reagent in which all the components are already
mixed in an optimized way. Additional stabilizers are added to enable long-term storage.
The concentration of components is perfectly adapted to most of the assays. Consequently,
the 2 x mix guarantees a high reproducibility, ease of use, and time saving.

Role of Core kits and MasterMixes components

Slight component changes can significantly impact your assay. It is therefore important to
consider them individually, understanding the role of each of them.

Taq Polymerase

A HotStart Taq polymerase is inactive at low temperatures (room temperature). Heating at

95 °C for several - usually 5 to 10 - minutes activates the enzyme, and the amplification
can begin once the primers are annealed. The enzyme is not active until the entire DNA is
denatured.

Two major HotStart modifications exist, the antibody-blocked Taq and the chemically-
blocked Taq. The antibody-blocked Taq is inactive because it is bound to a thermolabile
inhibitor that is denatured during the initial step of PCR. The chemically-blocked Taq
provides one clear advantage over the antibody-blocked Taq, as it is completely inactive
at 60 °C, (the hybridization temperature of primers), thus preventing the formation of non-
specific amplification and reducing primer dimer formation.

dNTPs / dUTPs

Some kits contain a blend of dNTPs and dUTPs, other ones contain only dNTPs. Using only
dNTPs increases the sensitivity, the reason being that the Taq incorporates more easily dNTPs
than dUTPs. However, using a mix containing dUTPs brings security to the assay, in case of
contamination from a previous PCR product. Thanks to the UNG activity in association with
incorporated dUTPs, this contamination can be eliminated.

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UNG

The Uracil-N-Glycosylase is an enzyme that hydrolyses all single-stranded and

double-stranded DNA containing dUTPs. Consequently, if all PCR amplifications are
performed in the presence of a dNTPs/dUTPs blend, by carrying a UNG step before every
run it is possible to get rid of any previous PCR product.

ROX

Some thermocyclers require MasterMix containing ROX dye for normalization. This is the
case for the ABI and Eppendorf machines, and optional on the Stratagene machines.
If you work with such machines, it is easier to work with the ROX dye already incorporated in
the MasterMix rather than adding it manually. It guarantees a higher level of reproducibility
and homogeneity of your assays.

Fluorescein

For iCycler iQ®, My iQ® and iQ5 machines (BioRad thermocyclers), the normalization
method for SYBR® Green assay uses Fluorescein to create a “virtual background”. As in
the case for the ROX, it is better and easier to use a MasterMix that contains pre-diluted
Fluorescein, guaranteeing higher reproducibility and homogeneity of your assays.

MgCl2

MgCl2 is necessary for the Reverse Transcriptase and the Taq activity. MgCl2 concentration
in MasterMixes is optimized according to the amount of Taq and also the buffer composition.
However, it may be necessary sometimes to add MgCl2 and most MasterMixes include an
additional tube of MgCl2.

Inert colored dye

Some buffers also include an inert colored dye, to enable visualization of the buffer when
loading in the wells. This colored dye has no effect on the sensitivity of the assay and is a
convenient working tool. Note that such mixes, in combination with white plastic plates,
provide better levels of fluorescence and a really easy way of working.

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 qPCR guide

Probe and primer quality

We really cannot insist enough, about the importance of this parameter. Your design is actually
the most variable parameter, especially in the case of SYBR® Green assays. It is not uncommon
to observe undetectable targets, high backgrounds or bad PCR efficiency, due only to the poor
design of the primers and probes. The next chapter details more precisely, the rules for probe
and primer design. However, consider also the quality and the purification level of your primers
and probes, as this can also influence your results.

The classical way of synthesizing oligonucleotides, is the standard Beta-cyanoethyl synthesis

cycle that includes: deblocking the first nucleotide, coupling of the second one, capping of
any previous nucleotide that has not been coupled, oxidation to stabilize the growing chain,
deblocking, then a new cycle starts. At the end of the oligonucleotide synthesis, the crude
product is cleaved from the solid support (CPG or polystyrene beads).

Concerning probe coupling, there are different methods by which a dye can be incorporated
into a probe. The choice of the coupling method used depends on the structure of the dye and
of the length of the probe.

Manual coupling
Manual coupling can be done via amine or thiol groups, using activated dyes such as Texas
Red®, TAMRA, JOE, Rhodamine (R6G) or ROX, BODIPY® and other dyes (Alexa®, Marina Blue®,
etc.). These labels can be linked internally to any dT-residue, dR-residue or to either terminus.
The fluorescent molecule is linked to the oligonucleotide via a spacer (a C-6 spacer generally)
to limit the steric hindrances.

Automatic coupling
Automatic coupling uses the appropriate phosphoramidite to which the dye is already coupled
like Cy® dyes, FAM, Fluorescein, HEX, TET and Yakima Yellow®. These labels are coupled during
the synthesis at the 5’ terminus.
The easiest probes to synthesize and to purify are Double-Dye Oligonucleotide probes, as they
are not too long. Moreover, the probes where you can use phosphoramidites (automatic coupling
method) are usually the easiest ones to synthesize and purify, as there is only one set-up for the
synthesis and one purification step. They also give the highest yields. The probes, which require
post-labelling procedures (manual coupling method) are more laborious to synthesize and will
generally yield lower amounts of product.

Purification level
Regarding the quality, it is recommended to work with purified primers for qPCR assays
(Cartridge Reverse Phase purified or HPLC purified primers are ideal). The first aim of any
purification method is to remove the by-products, resulting from the synthesis step (mainly
salts). The next goal is to enrich the product in full-length oligonucleotides. Consequently,
purified primers are more adapted to the sensitive qPCR tool than simply desalted primers. It is
particularly true when working with non-specific detection systems like SYBR® Green.

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Guidelines for primers and probes design

General rules
Well-designed primers and probes are a prerequisite for successful qRT-PCR. By using
well-designed primers and probes, PCR efficiencies of 100 % can be obtained.
If the following primer and probe design guidelines are taken into account you will achieve
high PCR efficiencies, specific PCR products, non co-amplification of gDNA and therefore
the most sensitive results.

We do recommend in general using a design software (for example Oligo® Primer Analysis
Software) to check for all following criteria.
Most thermocycler softwares now offer tools to help you designing primers with the best
characteristics. Some of the best softwares are Beacon Designer, Primer Express, and
DNA Star… Some other tools are freely available on the web, for example:

• http://medgen.ugent.be/rtprimerdb/ (human primer and probe database)

• http://frontend.bioinfo.rpi.edu/applications/mfold/ (for testing secondary structures)
• http://www.ebi.ac.uk/~lenov/meltinghome.html (Tm calculators)
• http://frodo.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
(Design Primer, also hybridization probes)
• http://bibiserv.techfak.uni-bielefeld.de/genefisher2
(Design Primer including degenerate primers)
• http://www.premierbiosoft.com/qpcr/index

Primers or SNP databases are also freely available, for example OMIM (from the NCBI), SNPedia,
Huge Navigator and RT Primer DB (Pattyn F. et al., 2006)

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 qPCR guide

Design guidelines for SYBR® Green I assays

As SYBR® Green I binds to any dsDNA it is important to avoid primer-dimers and non-specific
products in SYBR® Green I assays. This can only be avoided by carefully selecting primers that
only bind to the selected target. By selecting amplicons between 100 and 150 bp a high level of
fluorescence can be obtained without compromising the PCR efficiency.
One important characteristic of primers and probe is the Tm (melting temperature), the
temperature at which 50 % of the oligonucleotide is hybridized. The simplified calculation
method for the Tm is Tm=2(number A+T)+4(number G+C).

Primers

• Length : 18-30 bases

• GC content : 30-80 % (ideally 40-60 %)
• Tm : 55-60 °C, ∆Tm difference between forward primer and reverse primer should
be ≤ 4 °C
• Avoid mismatches between primers and target, especially towards the 3’ end of
the primer. Avoid runs of identical nucleotides, especially of 3 or more Gs or Cs
at the 3’ end.
• Avoid 3’ end T (allows mismatching)
• Avoid complementarities within the primers to avoid hairpins
• Avoid complementarities between the primers to avoid primer dimers, especially
of 2 or more bases at the 3’ ends of the primers
• Check if primers are unique and specific (check with a BLAST search:
www.ncbi.nlm.nih.gov/BLAST/)
• Design intron spanning or intron flanking primers to prevent amplification of
contaminating genomic DNA.
For intron spanning primers the first half of the oligo must hybridize to the 3’ end of one exon
and to the 5’ end of the other exon. In this way only cDNA will be amplified and not gDNA
(fig. 11).

Intron spanning primers

Forward
Reverse

gDNA

No amplification

Forward Reverse

cDNA

Amplification

Figure 11. Intron spanning primers

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For intron flanking primers, the forward primer must hybridize to one exon and the reverse primer
to the other exon. Amplicons from cDNA, without introns, will be smaller than the amplicons
from gDNA, which will contain the intron. The bigger amplicon will be amplified less efficiently.
The difference in size of these amplicons can be determined via meltcurve analysis. If genes of
only one exon are studied contamination with gDNA can only be avoided by DNase treatment
of the RNA with RNase free DNase (Vandesompele, 2002).

A recent study indicates that primers with a 5’ flap improve Real-Time PCR results
(Afonina, 2007). A unique validated 12-bases long AT-rich sequence added to the 5’ extremity of
both primers might increase the level of fluorescence and improve the sensitivity of the assay.
The addition of this sequence is claimed to be especially useful for short primers (when the
design is constrained) or on damaged DNA (for example sodium bisulfite treated DNA). The
positive effect has been observed both for SYBR® Green I and Double-Dye probe assays.

Amplicon

• Length : 80-150 bp
Shorter amplicons will give higher PCR efficiencies
Longer amplicons will give a higher ∆Rn as more SYBR® green I is incorporated
• GC content
30-80 % (ideally 40-60 %)
• Avoid secondary structures in the amplicon (check with Mfold: http://frontend.
bioinfo.rpi.edu/applications/mfold/).

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 qPCR guide

Design guidelines for Double-Dye probe assays

In probe assays, primer dimers and non-specific products will not be detected, however, they
will influence the PCR dynamics and efficiency.
Therefore, in probe assays they should be avoided as much as possible. For probe assays the
amplicons should be kept as short as possible, with the 5’ end of the probe as close as possible
to the 3’ end of the forward primer in case the probe is on the same strand, and as close as
possible to the 3’ end of the reverse primer in case the probe is on the opposite strand. In this
way the 5’ nuclease reaction will be optimal.
Experience has showed that it is easier to first design the probe, and then the primers, rather
than the other way around.

Primers and probes

Primers

Design rules for a probe assay are the same than for the SYBR® Green assay, see above.

Probes

• Length 18-30 bases, optimal length: 20

Lengths over 30 bases are possible, but it is recommended to position the quencher
not at the 3’ end, but internally 18-25 bases from the 5’ end (normally coupled to a T)
• GC content : 30-80 %
• Tm of the probe must be 8-10 °C higher than the Tm of the primers (8 °C for genotyping,
10 °C for expression profiling)
• Select the strand that gives the probe more Cs than Gs
• Place the probe as close as possible to the primers without overlapping them
• Avoid mismatch between probe and target
• Avoid complementarities within the probe
• Avoid runs of identical nucleotides, especially of 4 or more Gs
• Avoid 5’ end G (as this can quenche several fluorophores, including FAM)

For multiplex assays / genotyping

• Position the polymorphism in the center of the probe

• Adjust the probe length so that both probes have the same Tm

Amplicon
• Length
80-120 bp optimal
Shorter amplicons will give higher PCR efficiencies and more efficient 5’ nuclease
reactions
• GC content : 30-80 % (ideally 40-60 %)
Avoid secondary structures in the amplicon (check with Mfold:
http://frontend.bioinfo.rpi.edu/applications/mfold).

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Quenching principle
Fluorophore and quencher: definitions
Many commonly employed techniques for the detection of nucleic acid sequences in a
homogenous manner use fluorescence as the signaling technology. Typically a single-stranded
probe is labelled with a fluorophore and a quencher molecule. Changes in quenching of the
fluorophore, caused by hybridization of the probe to its target nucleic acid, lead to signal generation.

Thus, Real-Time PCR relies not only on the choice of one technology, but also in the choice of
the right fluorophore-quencher pairs, whether the assay would be singleplex or multiplex. The
choice of a fluorophore and its combination with a quencher, will give different results in terms
of sensitivity of the assay.
• A fluorophore is a molecule that emits light of a certain wavelength after having first
absorbed light of a specific but shorter wavelength. The emission wavelength is always
higher than the absorption wavelength.
• A quencher is a molecule that accepts energy from a fluorophore in the form of light and
dissipates this energy either in the form of light or heat.

Different ways of quenching

Quenchers are molecules that can accept energy from a fluorophore and dissipate it by two
mechanisms, called proximal, and FRET quenching. A fluorophore absorbs light energy and is
promoted to an excited state. In the absence of a quencher the fluorophore falls back to the
ground state and the excess of energy is released as fluorescence.

Figure 12. Fluorescence principle

Proximal quenching

When the fluorophore is in close proximity of a quencher molecule, the energy is transferred from
the fluorophore to the quencher, which then dissipates the energy as heat (no fluorescence is
observed). It is also known as collisional quenching. This type of quenching is used in Molecular
Beacons and Scorpions® primers.

Figure 13. Proximal quenching principle

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 qPCR guide

FRET quenching
The fluorophore transfers its energy to the quencher (which may be another fluorophore); the
energy is released from the quencher as fluorescence of a higher wavelength. The efficiency of
this process is dependent on the distance between fluorophore and quencher, more precisely
on the Förster distance 1/r6 (where “r„ is the fluorophore – quencher distance). This type of
quenching is used in Double-Dyes probes and LC Hybridization probes. Dark quenchers
dissipate the energy as heat, resulting in an absence of fluorescence emitted by the quencher.
This type of quenchers allows a better signal-to-noise ratio in the assay.

Figure 14. FRET quenching principle

Optimal fluorophore quencher combination

The quencher has to fit the fluorophore
One fluorophore can be combined with multiple different quenchers, but the absorption spectrum
of the quencher needs to have good overlap with the emission spectrum of the fluorophore in
order to achieve optimal quenching.

• For example, Deep Dark Quencher II absorbs over a large range of the visible
spectrum and therefore efficiently quenches most of the commonly used
fluorophores, especially those emitting at higher wavelengths.
• Deep Dark Quencher I and Eclipse® Dark Quencher, quench the lower wavelength
dyes such as FAM but they are not good at quenching those that emit at high
wavelengths such as Cy® dyes.
• The Black Hole Quencher family covers a large range of wavelengths (over the
entire visible spectrum and into the near-IR) and is consequently one of the best
options for multiplex application.

Quenchers have a quenching capacity throughout their absorption spectrum, but the
performance is best close to the absorption maximum.

Quencher Absorption (nm) Emission (nm)

TAMRA 565 580
BHQ-1™ 534 none
BHQ-2™ 580 none
BHQ-3™
670 none
Deep Dark Quencher 1 430 none
Deep Dark Quencher 1 630 none
Dabcyl 475 none

Table 3: Absorbance and emission wavelengths of common Quenchers ( BHQ: Black Hole Quencher)

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 33

4 6 Emission maxima
3 of common dyes:
1 5
1. FAM
2
0.08 2. Yakima Yellow® - VIC®
Absorbance

3. TAMRA
4. CY®3
0.06
5. ROX
6. CY®5

0.04

0.02

0
400 500 600 700
Wavelength (nm)

Figure 15. UV-vis absorbance spectra of Quencher

Deep Dark Deep I Dark Quencher (pink),
Eclipse® Dark QuencherDabCYL (grey), Eclipse ® Dark Quencher
(purple) and Deep Dark Quencher IIDABCYL
(orange) Deep Dark Quencher II

23 6 7 9 10 Emission maxima
Absorbance

of common dyes
5
1 4 8
1. FAM
2. TET
3. Yakima Yellow®
4. HEX
5. Cy®3
6. TAMRA
7. Cy®3.5
8. ROX

500 600 700 9. Cy®5

10. Cy®5.5
Wavelength (nm)
BHQ-0TM
BHQ-1 TM
BHQ-2 TM
BHQ-3 TM

Figure 16. UV absorbance spectra of the Black Hole Quencher™ family

Figure 17. Absorbance range of Dark Quenchers and emission maxima for Reporter Dyes

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 qPCR guide

Dye-thermocyclers compatibility table

Almost all detection systems work on almost all Real-Time thermocyclers. The detection
channels determine which fluorophores have to be used, and the combination of excitation and
detection channels determine which probe systems can be used. It is important to consider,
which dyes are the best for multiplexing on the particular machine being used and also any
requirements for spectral calibration. This is required because dyes exhibit some spectral
overlap, making it necessary to calibrate the machine, which is able to distinguish between the
dyes. Spectral calibration kits can be bought for the majority of machines where this may be
required.

Double-Dye Oligonucleotides in single-color detection

For single-color detection we recommend to use the combination FAM-TAMRA,
FAM-Eclipse® Dark Quencher or FAM-Black Hole Quencher 1. These combinations are the
most standard ones, they can be detected on all Real-Time PCR thermocyclers and are
easy to synthesize.

Double-Dye Oligonucleotides for multiplexing detection

For multiplex Real-Time PCR, it is very important to select dyes that give a good spectral
separation to avoid overlap of the signal. For the first color we recommend the use of
FAM-Black Hole Quencher 1. This is the best choice for duplexing or multiplexing PCR, as
BHQ-1™ gives a lower background compared to TAMRA.

For the second color, we recommend the use of Yakima Yellow® - BHQ-1™. Yakima Yellow®
gives a good spectral separation from FAM, so is the best choice to be combined to
FAM-BHQ-1™. Furthermore, it is a good cost effective alternative to VIC, and it can be detected
in the same channel at the same wavelength, with no additional calibration needed.

The choice of the third color is dependent on the thermocycler (Table 4). We recommend
using either DFO (Dragon Fly Orange), or Texas Red®. These two dyes have both a good
spectral separation from FAM and Yakima Yellow®, and both give efficient synthesis with
high yields. The choice of the fourth dye is also dependent on the Real-Time thermocycler.
We usually recommend the use of Cy® 5, as Cy® 5 has a good spectral separation from
FAM, Yakima Yellow® and Texas Red®. Furthermore, this last one also has an efficient
synthesis and gives high yields.

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 Thermocycler Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 Channel 7
GeneAmp® SDS 5700 FAM

ABI Prism® SDS 7000 FAM YY/VIC/JOE DFO/NED ROX −

ABI Prism® SDS 7700 FAM YY/VIC/TET/JOE DFO/NED ROX −

ABI Prism® SDS 7900 HT FAM YY/VIC/TET/JOE DFO/NED ROX −

ABI Prism® SDS 7300 FAM YY/VIC/JOE DFO/NED ROX −

ABI Prism® SDS 7500 FAM YY/VIC/JOE DFO/NED/Cy®3 ROX/TR Cy®5 −

StepOne® FAM YY/VIC/JOE ROX − −

StepOnePlus® FAM YY/VIC/JOE DFO/NED ROX −

Cycler iQ® FAM YY/VIC/HEX/TET/ Cy®3 DFO/NED ROX Cy®5

My iQ® FAM −

Table 4. Dye-thermocycler compatibility table.

iQ5 FAM YY/VIC/HEX/TET DFO/NED ROX/TR Cy®5 −
® ®
CFX 96 FAM YY/VIC/HEX ROX / TR Cy 5 Cy®5.5 / Quasar 705

MiniOpticon® FAM YY/VIC/HEX/TET − − −

DNA Engine Opticon® 1 FAM − − −

DNA Engine Opticon® 2 FAM YY/VIC/HEX/TET − − −

Chromo 4® FAM YY/VIC/JOE/TET DFO/NED/ROX/TR Cy®5 −

® ®
Mx3000P (choice of 4 filtres) FAM TET YY/VIC/JOE/HEX Cy 3 DFO/NED TR/ROX Cy®5
® ®
Mx3005P (choice of 5 filtres) FAM TET YY/VIC/JOE/HEX Cy 3 DFO/NED TR/ROX Cy®5

Mx4000® (choice of 4 filtres) FAM TET YY/VIC/JOE/HEX Cy®3 DFO/NED TR/ROX Cy®5

Mastercycler®ep realplex2 FAM YY/VIC/JOE/HEX/TET − − −

Mastercycler®ep realplex4 FAM YY/VIC/JOE/HEX/TET DFO/TAMRA ROX −

LightCycler® 1.5 FAM YY/VIC/JOE/HEX/TET LC Red 705/ Cy®5 −

LightCycler® 2 FAM LC Red 640/ROX LC Red 610 LC Red 640 LC Red 670 LC Red 705

LightCycler 480® FAM YY/VIC/HEX LC Red 610 LC Red 640/ROX Cy®5 LC Cyan 500

Smartcycler®1 FAM YY/VIC/TET/JOE/ Cy®3 TR Cy®5

Smartcycler®2 FAM Cy®3/ TET TR/ROX Cy®5

35

Rotor-Gene™ 2000 / 3000 FAM YY/VIC/JOE/TET ROX/Cy®3.5 Cy®5

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Rotor-Gene™ 6000 FAM YY/VIC/JOE/TET/HEX ROX/TR/Cy®3.5 Cy®5 Alexa®680/Atto680 Alexa® Fluor-350

YY: Yakima Yellow® TR: Texas Red® DFO: DragonFly Orange®

 qPCR guide

Example of a classical assay

Some tips and tricks before starting

• Don’t forget to include a negative control, and eventually an external or internal positive
control (IPC). Choose your normalization method (absolute or relative quantification),
cf. p. 39.
• Prepare carefully your plate layout, to avoid any doubt while pipetting. No Template
Controls should be placed on the plate in such a position that cross contamination
is avoided during the set-up; thus, they should be placed if possible away from the
highest DNA concentrations, at the top or bottom of the rows, to avoid spilling over the
wells, when pipetting.
• Defrost all reagents on ice, and keep SYBR® Green® mix and probes away from light.
• Prepare the reaction mix ideally in a separate room, different from the room where the
DNA samples have been prepared, to avoid any contamination.
• Prepare a reaction mix with a volume 5 to 10 % higher than the volume needed. Actually,
some pipetting volumes are very small and small droplets can remain outside the tips,
leading to loss of reagents. Working in this way can avoid a lack of mix at the end of
the plate preparation!
• Don’t wait too long after the plate preparation. It is always better to run the qPCR
straight after the preparation. You might need to compare two identical plates/samples
with different cycling conditions. In this case only, it is better to prepare plates at the
same time, then to store one of them in the fridge at 4 °C away from light. You can keep
it like this for up to 10 hours.
• When sealing the PCR plates, it is important to ensure that it has been correctly done. If
optical films are used, fingerprints and marks should be avoided on the top of the film.
Pay special attention to stick the lids to the side of the plate, to avoid evaporation.
• Pipette your sample in triplicate or at least duplicate.
• The plate can be shaken on a plate shaker and spun, before placing it on the machine.
This is not an essential step but will ensure that the reaction components are thoroughly
mixed and collected at the bottom of the reaction tube. It is useful to also check the
wells for bubbles, as bubbles at the bottom of the well can produce unusual plots on
the results (when using machines that read from the top).

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 37

Classical protocol and thermal profile

By respecting the tips above, prepare a reaction mix containing primers, probes
(if applicable), and all the reagents required for the reaction. It is always better to proceed
like this because it minimizes differences across the plate and allows for more accurate
pipetting.

Commercial MasterMixes often are 2x MasterMixes, meaning half of your reaction volume is
MasterMix, and half is composed of primers, probe and template. The primers final concentration
should be between 100 nM and 300 nM for a SYBR® Green I assay, and between 100 nM
and 900 nM for a probe assay. Probe concentration should be between 100 nM and 300 nM.
Nowadays, usual final volume is 25 µl, however it is often possible to work in 20 µl or less to
save reagents costs.

Add the DNA sample (template) on the side of each well (water for the NTC), before the reaction
mix as it will be transferred down when adding the reaction mix. As the reaction mix is heavier
than the DNA, the DNA will be mixed within the reaction mix.

Then, run a first qPCR with the classical thermal cycles on Figure 18.

Thermal Profile

100
95° 95° 1
END
10:00 00:15
75
Temperature

01:00
02:00 60°
50 50°

1 2 3 4
25

0
Segment 1 Segment 2 Segment 3
1 Cycle 1 Cycle 45 Cycles

Figure 18. Classical thermal profile including a UNG activation step carry-over prevention step

1. The first step at 50 °C is the UNG step. It is not necessary with all the reagents, as some of
them do not include UNG. During this step, the UNG cleaves any contaminating template
containing U bases.

2. The 10 minute-step at 95 °C activates the Taq and denatures the UNG. Some Taq may
require shorter activation steps.

3. 15 seconds at 95 °C is the first step of the repeated PCR cycles. The dsDNA template
denatures at this temperature.

4. 60 °C for 1 minute, allows the annealing and extension of the primers by the Taq.

Depending on your thermocycler, and the characteristics of your primers, the protocol can be
shortened and the temperatures adapted. Our optimization tips (p. 49) can help you adapt it to
your assay.

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 qPCR guide

Useful definitions
To understand the first results graphs you get, it is necessary to be familiar with some definitions
and concepts.

The Amplification Curve or Primary Growth Curve is usually the first graph we look at. It
shows the increase of fluorescence level (called Rn, Rfu, F1…) on the Y axis, compared to the
run cycle number on the X axis.

Figure 19. Threshold definition and Ct value calculation

The baseline is the average background. It is calculated according to the noise level in the early
cycles, when there is no detectable increase in fluorescence, due to PCR products. When the
baseline is set up manually, it is important to ensure that only cycles where there is no signal
increase are selected !

The threshold is the level of fluorescence above the baseline, at which the signal can be
considered not to be background. The threshold can be calculated automatically or can be set
manually. The automatic calculation of the threshold corresponds to the average baseline + “X”
standard deviation of this baseline.

The Ct value (Cp for Roche’s thermocyclers) is defined as the cycle in which there is a significant
increase in reporter signal, above the threshold, i.e. the cycle in which the growth curve crosses
the threshold. It is consequently related to the initial amount of DNA and shows also the
sensitivity of the assay. The Ct value is consequently in inverse proportion to the expression
level of the gene. If the Ct value is low, it means the fluorescence crosses the threshold early,
meaning that the amount of target in the sample is high. The Ct is therefore dependent on the
threshold level, and it is then important to compare the threshold value from one run to another,
if no normalization method is used.

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 39

The ROX is a fluorescent dye that is used as a passive reference for some thermocyclers
(Eppendorf, ABI Prism®, Stratagene…). This dye is usually spiked into the MasterMix and the
reporter fluorescence is normalized to the ROX signal on ABI and Stratagene Mx machines.

The Rn (normalized reporter signal) is calculated by dividing the reporter signal by the ROX
signal. This signal is then corrected for pipetting inaccuracies, variation in sample volume,
bubbles in sample, plastics inconsistency…

When correction for background variations is required, the ∆Rn is used. The ∆Rn is calculated
by subtracting the normalized baseline from the normalized reporter signal.

∆Rn = Rn-Bn where:

Rn= (reporter signal / ROX passive reference signal)

Bn= (baseline signal / ROX passive reference)

Only a minor variation in the ROX passive reference will cause a change in the Rn, Bn, ∆Rn,
threshold and Ct value. Therefore we recommend using MasterMixes that already contain a
ROX passive reference to avoid such variations, which might be interpreted as real differences,
but in reality are just experimental artifacts.

Normalization and quantification methods

When analyzing and comparing results of Real-Time qPCR assays many researchers are
confronted with several uncontrolled variables, which can lead to misinterpretation of the results.
Those uncontrolled variables can be the amount of starting material, enzymatic efficiencies, and
differences between tissues, individuals or experimental conditions.

In order to make a good comparison, normalization can be used as a correction method, for
these variables.

There are several ways to normalize, which all have some advantages and disadvantages.

The most commonly known and used ways of normalization are : normalization to the original
number of cells, normalization to the total RNA mass, normalization to one or more housekeeping
genes, and normalization to an internal or external calibrator.

Normalization to number of cells can actually only be done for cell culture and blood samples. In
solid tissues and tumors, the amount of cells cannot be counted, only estimated and therefore
leads to inaccuracies (Bustin, 2000).

Normalizing to the RNA mass quantity will lead to inaccuracies as the total RNA mass (rRNA,
tRNA and mRNA) might contain varying imbalances between rRNA and mRNA (Solinas et al.,
2001). The determination of the RNA amount by photospectrometry is another factor causing
inaccuracy, as the A260 quantification is strongly influenced by the impurity of the sample,
caused by contaminating DNA, free nucleotides and proteins (Bustin, 2000 – Bustin, 2002). As
very little is known about total RNA content per cell (in different tissues), a variation between
individuals and between normal or highly proliferating tissues, like tumors will cause unwanted
inaccuracies (Bustin, 2000 – Bustin, 2002).

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 qPCR guide

Normalization to one housekeeping gene excludes some of the above-mentioned drawbacks,

but is also not the perfect method. The advantage is that the variation due to different amounts
of RNA can be excluded. However, one must assure oneself, that the housekeeping gene is
expressed constantly at the same level throughout the experiment and between samples. It is
better then to use several housekeeping genes. A housekeeping gene used for normalization
can also be used as a control (cf. p. 45).

The two majors methods of normalization are the absolute quantification and the relative
quantification (Sellars MJ et al., 2007). Figure 20 summarizes the quantification methods that
will be developed further.

In absolute quantification, the exact number of copies of the gene of interest is calculated. In
relative quantification, the expression of the gene of interest in a sample is expressed relatively
to another gene, another sample, used as a reference.

Absolute quantification Relative quantification

External calibration External calibration

curve curve
External calibration
/PSNBMJ[BUJPO
XJUI
POF
PS
NVMUJQMF
4:#3
(SFFO
BTTBZ 1SPCFT
BTTBZT DVSWF
XJUIPVU
BOZ
SFGFSFODF
HFOFT
with or without ROX with or without ROX SFGFSFODF
HFOF
OPSNBMJ[BUJPO OPSNBMJ[BUJPO

Standard curve with: 8JUIPVU
3FBM
5JNF
1$3 8JUI
1$3
FîDJFODZ

FîDJFODZ
DPSSFDUJPO correction
r
351$3
QSPEVDU ΔΔ$U
NFUIPE TFF
GPSNVMB
JO
UIF
UFYU
r
TZOUIFUJD
3/"
r
TZOUIFUJD
3/"
r
TZOUIFUJD
%/"
r
QMBTNJE
r
QSFWJPVT
1$3
QSPEVDU
r
QPPMFE
D%/"
r
H%/"

Figure 20: Summary of quantification methods in Real-Time qPCR

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 41

Absolute quantification
Absolute quantification requires a standard curve of known copy numbers (cf. p. 46 for the
construction and analysis of the standard curve). It can be constructed using several standards
(Figure 21).

Plasmid, linear
PCR product
Pooled cDNA
Genomic DNA
Commercial RNA
Standard

In Vitro Trans RNA

None
Plasmid, not linear
Other
Bacteria
Plasma
Plasmid + Stds

0 10 20 30 40 50 60

Figure 21: Most frequently used quantification standards. From Nucleic Acid Research Group, (NARG) survey 2007,
http://www.abrf.org/NARG/

The amplicon being studied can be cloned, or a synthetic oligonucleotide (RNA or DNA) can
be used. For gene expression studies, it is recommended to use an RNA standard, so the
quantification method takes into consideration any possible RT efficiency variation.

The standard must be amplified using the same primers as the gene of interest and must amplify
with the same efficiency. The standards must also be quantified accurately. This can be carried
out by reading the absorbance at A260, although this does not distinguish between DNA and
RNA, or by using a fluorescent ribonucleic acid stain such as RiboGreen. The problem when
using in vitro transcribed RNA as standard is that the construction of cDNA plasmid has to be
done and has to be reverse transcribed. Moreover, it is not very stable for long-term storage.
The advantages of using cDNA plasmid standards is that cDNA plasmids are easy to construct,
they can be prepared in large amount and can be stored for a very long time without any
difficulties.

Relative quantification
Relative quantification is the most widely used technique. Gene expression levels are calculated
by the ratio between the amount of target gene and an endogenous reference gene, which is
present in all samples. The reference gene has to be chosen so that its expression does not
change under the experimental conditions or between different tissues (Cook NL et al., 2008).
There are simple and more complex methods for relative quantification, depending on the PCR
efficiency, and the number of reference genes used.

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 qPCR guide

Delta delta Ct (∆∆Ct) method

This method is the simplest one, as it is a direct comparison of Ct values between the target gene
and the reference gene. However, PCR efficiencies of both the target and of the reference gene
should be close to 100 % and not differ by more than 10 %. Only the initial experiment requires
a standard curve to compare the PCR efficiency of the target and control gene (Schmittgen TD,
et al., 2008).

Relative quantification involves the choice of a calibrator sample. The calibrator sample can be
the untreated sample, the time=0 sample, or any sample you want to compare your unknown to.

Firstly, the ∆Ct between the target gene and the reference gene is calculated for each sample
(for the unknown samples and also for the calibrator sample).

∆Ct = Cttarget – Ctreference gene

Then the difference between the ∆Ct of the unknown and the ∆Ct of the calibrator is calculated,
giving the ∆∆Ct value:

∆∆Ct = (Cttarget – Ctreference)calibrator – (Cttarget – Ctreference)sample

The normalized target amount in the sample is then equal to 2-∆∆Ct and this value can be used to
compare expression levels in samples.

Different PCR efficiencies

If there is more than 10 % difference in PCR efficiencies, between the reference gene and the
target gene, then it is inaccurate to use the ∆∆Ct method. The value used is then:

Ratio = (Etarget) ∆Ct target (calibrator - sample) / (Ereference) ∆Ct reference (calibrator - sample) where :

Etarget = PCR efficiency of the target

∆Ct target (calibrator - sample) = (Cttarget) in the calibrator - (Cttarget) in the sample.

When several reference genes are used

As mentioned in the Normalization chapter, it is better to use several control genes, because
the expression of one reference gene might change slightly. It is then necessary to use an index
reference gene (REF), a method described by M. Pfaffl and J. Vandesompele, 2004.

The normalization by geometrical averaging of multiple internal control genes (Vandesompele

et al., 2002) is a robust and innovative approach for accurate normalization. It relies on the use
of several housekeeping genes to even out variations in the expression of these genes.

Only the most stable expressed housekeeping genes are taken into the calculation.
The calculation can be performed using the freely available software geNorm
(http://medgen.ugent.be/~jvdesomp/genorm/). The major drawback of this method is the need
for many primer pairs and the complicated way to process the data.

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 43

Evaluation of your qPCR assay

Several parameters can help you validating your assay. As for any experiment, before using a
qPCR test routinely, it is important to optimize it, and validate its reproducibility.

Controls
The optimal way of processing is to include several negative and positive controls. The main
ones are shown on Figure 22.

No Template Control (NTC) to check for contamination

Biological replicate (ie., different samples, same treatment)

Standard curve
Control/Replicate

Technical replicate-same cDNA sample following RT reaction

Minus RT or RNA control to check for genomic DNA contamination

External (exogeneous) positive control

Technical replicate-same RNA sample prior to RT reaction

External (exogeneous) negative control

Internal Positive Control (IPC) to check for PCR inhibition

None / Not applicable

1 2 3 4 5

Frequency (1=Least, 5=Most)

Figure 22: Frequency of replicates controls. From Nucleic Acid Research Group, NARG survey 2007,
http://www.abrf.org/NARG/

Negative Controls
• NTC: the No Template Control contains water and reaction mix, instead of
template and reaction mix. Of course you should not observe any amplification in
the NTC. However, it is sometimes difficult to achieve. In SYBR® Green assays,
if you have many constraints on the design of the primers, you might observe a
late amplification due to primer dimers. In any case, the amplification in the non-
template control should go up late, and be at least 8 Ct after your specific signal,
thus avoiding false positive interpretation. If it is not possible to achieve such a
result, it is better to switch to a probe assay that is more specific. A minimum of
2 NTC’s should be included.
• NAC: the No Amplification Control includes all the components except the
Polymerase. It is not possible to do one if you use a MasterMix that contains
everything already. However, you can add only probe, primers, template and water,
to check the integrity of the probe. If you observe an increase of fluorescence in
this case, it is most likely that the probe is degraded. The NAC is less important
than the other negative controls, as the probe degradation might also be detected
with a high background level.
• No RT: the No Reverse Transcriptase control is a sample without the addition of
the Reverse transcriptase prior to the PCR step. If the No RT control shows an
amplification (and not the NTC control), you can conclude that contaminating genomic
DNA is present. The best way to proceed then is to design intron spanning primers or
intron flanking primers.

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 qPCR guide

Positive controls
Their main goals are to detect the quality of the reagents used, the presence of inhibitors in your
sample, or damaged sample. Positive controls can also be used for normalization.

Endogenous Positive Control

An endogenous control is a second target for which the expression level is measured in the
same sample than the Gene of Interest (GOI=target). In can be run in multiplex or in a different
well. Endogenous positive control gives indication on the buffer or polymerase action.

An endogenous control gene can be used to standardize the amount of sample RNA or DNA
added to the reaction. It should be a stable expressed gene through the experiment, that is
used to normalize the results of a variable target gene, and to correct for sample-to-sample
variations.

Exogenous Positive Control

It is a different sample in which, the gene of interest is known to be expressed. It can be a

previous PCR product, a plasmid containing the gene of interest, in vitro RNA transcripts…
Thanks to this control, it is possible to check the quality of the primers, probe, and reagents but
won’t give any indication concerning the sample (degradation, inhibitors…).

Spiking Control or Internal Positive Control (IPC): it is a DNA or RNA that is spiked into the
sample and amplified using specific primers. Any variation of this spiking control expression
would show that inhibitors are present in the sample (Scipioni A. et al., 2008).

qPCR controls
NEGATIVE AIM
r
/P
5FNQMBUF
$POUSPM Detection of primers dimers and
contamination

r
/P
"NQMJñDBUJPO
$POUSPM Detection of probe’s degradation

r
/P
35
$POUSPM Detection of genomic DNA

contamination

POSITIVE AIM
r
&OEPHFOPVT
$POUSPM Check quality of reagents.
(same sample, different target) Also used for normalization.

r
&YPHFOPVT
$POUSPM Check quality of reagents.

(same target, different sample)

r
4QJLJOH
$POUSPM Detect inhibitors presence

(additional DNA spiked into the sample, 3FKFDU
GBMTF
OFHBUJWF
JO
EJBHOPTUJD
BTTBZT
different target)

Figure 23. Summary of the main existing controls in qPCR and their interest.

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 45

Commonly used control kits

A good control should have a constant level of expression between individuals, among different
tissues of an organism, at all stages of development, and should not be affected by the experiment
treatment. The control gene should also be expressed at similar level as the gene of interest
and the range of linear amplification should be known (Bustin, 2000). If a control gene is up and
down regulated by the experimental intervention, this will lead to an incorrect normalization and
thus to misinterpretation of the results. Therefore, proper validation of presumed stability of
expression of the control genes, should be done before studying the target gene.

The table below describes the expression level of commonly used control genes.

Relative
Symbol Accession number Name Pseudogene
expression level
- M10098 18S rRNA0 Very high

- M11167 28S rRNA0 Very high

ACTB NM_00101 Beta actin + High

GAPD NM_002046 Glyceraldehyde-3-phosphate dehydrogenase + High

UBC M26880 Ubiquitin C - High

B2M NM_003194 Beta-2-microglobulin - High

YWHAZ NM_000190 Tyrosine 3-monooxygenase activation protein, Zeta + Medium

polypeptide (Phospholipase A2)

RPL13A NM_012423 Ribosomal protein L13a + Medium

SDHA NM_004168 Succinate dehydrogenae complex, subunit A + Medium

HPRT1 NM_0000194 Hypoxanthine photshoribosyl-transferase I + Medium

TBP NM_003194 TAT box binding protein - Low

HMBS NM_000190 Hydroxymethyl-bilane synthase Low

Table 5. Relative expression level and presence (+) or absence (-) of pseudogenes in the genome concerning the
most commonly used control genes

Singleplex or multiplex assay for the controls?

The endogenous control can be quantified in a singleplex reaction. The gene of interest and
control are, in this case, amplified in separate reactions, using the same cDNA sample. It
can also be used in a multiplex reaction, which prevents pipetting errors between the control
reaction and gene of interest reaction, which can occur when using singleplex reactions. In this
case, normalizing in the same well, will account for the variability in individual PCR assays, like
inhibitors. Moreover, multiplex assays have the advantage of being cost effective in terms of
reagents required.

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 qPCR guide

Role of the standard curve and evaluation

of its parameters
Preparing a standard curve for each gene, which needs to be analysed, can provide a good
idea of the performance of the qPCR. The standard curve should cover the complete range
of expected expression. Using standard material (plasmid, PCR product etc…), the standard
curve should include at least 5 points of dilution, each of them in duplicate (at least). The 10-fold
or 2-fold dilution range should cover the largest range of expression levels. Plotting these
points on a standard curve, will determine the linearity, the efficiency, the sensitivity and the
reproducibility of your assay.

Standard Curve
Std. curve samples

21

20
Crossing Point

19

18

17

16

-1 0

Log Concentration

Figure 24: Example of a 2 times-serial diluted cDNA standard curve. Detection using SYBR® Green I on a LC480
thermocycler.

PCR efficiency
The slope of the standard curve gives the efficiency of the PCR reaction by the following
equations:

Exponential amplification = 10(-1/slope)

Efficiency = 10(-1/slope) –1

If the slope of the standard curve is -3.32 then the PCR is 100 % efficient.

With 100 % efficiency, a 2x dilution gives a ∆Ct of 1 between each dilution (each cycle the
amount of amplification is doubled).

With a 100 % efficiency, a 10x dilution gives a ∆Ct of 3.2 values between each dilution (every
3.2 cycles the amount of amplification is 10 fold higher).

PCR efficiency between 90 %-110 % is acceptable (i.e. a slope between 3.1 and 3.58)

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 47

The following factors influence the PCR efficiency:

• Length of the amplicon

• GC content of the amplicon
• Secondary structures in primers, and / or probe, and / or amplicon
• Concentration of reagents

R square (R²)
The R² is a parameter, which indicates how well the data points lie on line. It shows consequently
the linearity of the PCR assay. If the R² < 0.95 there is an indication that either the reactions have
not been pipetted accurately, or that there is no linear relation between the Ct and the 10 log of
the DNA concentration. The latter can be caused by inhibitory factors that are diluted out. A R²
higher than 0.985 is acceptable.

Sensitivity
You can compare the sensitivity of your assay by comparing the Ct values. The lower your Ct
value is, the higher your assay sensitivity. However, be careful when you compare an assay
in terms of sensitivity. You must compare it with the same sample, under the same running
conditions, using the same threshold level. A dispersion of the duplicates/triplicates at the lower
concentrated sample on the standard curve, can also indicate a lack of sensitivity.

Reproducibility
The reproducibility is indicated by the replicates. The standard deviation between the
replicates can be calculated. It’s ideally better to avoid replicates that have more than 0.5 Ct of
difference

Specificity of a SYBR® Green I assay

For SYBR® Green I assay, an additional point has to be taken into consideration for the
evaluation of the assay, this is the specificity. At the end of the run, it’s better to systematically
run a meltcurve. The temperature is slowly increased from 60 °C to 95 °C whilst continuously
monitoring the fluorescence. At a certain temperature, the whole amplified product will fully
dissociate, resulting in a drop of fluorescence as the SYBR® Green dissociates from the dsDNA.
As the temperature of dissociation is dependant on the length and composition of the amplicon,
it is consequently possible to check how many products of amplification are present in the
well. A nice meltcurve should show a unique dissociation peak (figure 25). If primer dimers are
amplified, a small product is usually observed around 70 °C (figure 26). The primer dimer peak
can sometimes even be higher than the peak of the specific product. The primer dimer peak
will also be observed even in the NTC control, reinforcing the conclusion that the amplification
is non-specific.

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 qPCR guide

Figure 25. Clean meltcurve (without any primers dimers)

3.0
2.8
2.6
2.4
2.2
Fluorescence -d(F1)/dT

2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
67.0 68.0 70.0 72.0 74.0 76.0 78.0 80.0 82.0 84.0 86.0 88.0 90.0 92.0 93.0

Temperature (°C)

Figure 26. Meltcurve with primers dimers (corresponding to the first peak around 74 °C)

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 49

Optimization tips

In general
If the PCR efficiency is drastically lower than 100 % we would recommend the following:

• Check the primer design and optimize the primer concentration

Trying a new design is sometimes the quickest and cheapest option. To optimize an
assay, it is also important to determine the best primer and probe concentration. We
recommend testing a matrix of primer and probe concentrations. For SYBR® Green
assay: test primers at 50 nM, 100 nM and 300 nM final concentration, each one
independently of the other. For probe assay, test primers at 100 nM, 300 nM and 900
nM and test probes at 100 nM and 250 nM. The best combination of forward primer,
reverse primer and probe concentrations can then be selected.

• Optimize the temperature reaction and times

Try to change annealing temperature. Start at 60 °C, and then try 58 °C, 56 °C, 54 °C.
Try also to increase it up to 64 °C. This annealing temperature of course depends of
the Tm of your primers.

• Go for a 3-step instead of a 2-step protocol

In the following cases we recommend performing a 3-step protocol to obtain better
results: if results show late Ct values, if the amplicon is quite long, if the results give a
less steep growth curve, or if it is not possible to re-do the design.

The protocol will then be as follows:

40 cycles Denaturation 95 °C for 15 seconds

Annealing 60 °C for 30 seconds

Extension 72 °C for 30 seconds

Extension time can be increased with 10-second steps.

It is also possible to add a data collection step after the extension step. To do so, ensure
that the detection temperature is at least 3 °C lower than the Tm of the amplicon.

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 qPCR guide

For probe multiplexing assay/qualitative qPCR

General way of processing for a multiplex assay:

• Check carefully the possible complementarity between probes and primers for the
different targets.
• Perform (with each target) a singleplex qPCR, with a standard curve to calculate
the PCR efficiency.
• Optimize individually each singleplex assays to reach a PCR efficiency as close to
100 %. Multiplex and calculate again the PCR efficiency and the sensitivity of
each target. You should not observe a big switch when comparing singleplex
and multiplex results for the same target. If there is a loss of sensitivity/efficiency
between the singleplex and the multiplex experiment, then try the following things,
in this order:
• Limit the probe/primer concentration of the most abundant target.
• Optimize the annealing temperatures by trying steps of 2 °C higher and lower than
the current annealing temperature.
• Increase the primer/probe concentration of the less abundant target.

It is actually important to do it in this order, as the main goal is to keep the total primers/
probe concentration the lowest possible. A high concentration could partially inhibit the Taq
polymerase activity.
Such problems can arise when amplification of both genes compete for the PCR reagents.
A highly abundant gene can out-compete a less abundant template leading to a bias in the
results.

Limiting the primer concentration for the most abundant template can avoid this competition.
However, this takes a considerable amount of development. Furthermore, even when working
with limited primer concentrations, the detection of rarer target genes may not be possible or
may be detectable at much reduced efficiency. Optimizing can also mean adding more units of
Taq polymerase per reaction and therefore increasing the costs again.

• Be more flexible with your design by using LNA® bases. By increasing the Tm of
the probe, LNA® probes size can be reduced (cf. p. 15) and specificity increased.
• Use a Dark Quencher: when multiplexing, the number of probes in the well
increases, and this can lead to an increase of basal fluorescence. Moreover,
quenchers such as TAMRA are also fluorophores, leading to high background
fluorescence. That is why we recommend the use of Dark Quencher for multiplex
assays. There are now several ‘Dark quenchers’ available. These absorb the energy
emitted by the fluorophore, but release it as heat rather than fluorescence, thus
leading to a better signal to noise ratio (cf. “Quenching principle”). Dark quenchers
are completely non-fluorescent, and have an extremely low background. Using
dark quenchers significantly improves signal to noise ratios (higher ∆Rn, a lower
background), and thus give a higher sensitivity in multiplex PCR.

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 51

Troubleshooting guide

Contamination

With genomic DNA

The best proof of contamination with genomic DNA is a clean NTC, and a No RT control showing
amplification. In this case, carry out a fresh extraction and RT step. If your sample is rare and
precious, then use intron spanning primers (cf. p. 28).

From a previous PCR run

When using MasterMixes containing a blend of dUTPs and dNTPs, you can easily get rid of a
contamination from a previous PCR run. Just use the UNG at the beginning of each run, this will
destroy any PCR product containing dUTPs.

Bad PCR efficiency due to primer dimers

Primer dimers in SYBR® Green I assays are easily identifiable on the meltcurve, and the
consequence would be a bad PCR efficiency. If you have the choice of the region in which you
can design your primers, then the best way to proceed is to do another design. The time wasted
for the achieved doubtful result during optimization of an assay with badly designed primers
costs much more than a new pair of primers.
If you are limited in the choice of the region for the design, then try to decrease the concentration
of primers. You can also try different MgCl2 concentrations. The best way to proceed however
would be to switch to a probe assay which can be more specific.

Inhibition
Decreasing the amount of template can identify the presence of PCR inhibitors in the template.
If the Ct values decrease when decreasing amounts of template are used (inverted result), then,
it is usually characteristic of the presence of inhibitors. Using an external spiking control can
also show the presence of inhibitors.

How to get rid of inhibitors?

There are now several commercial kits to purify RNA samples on columns. You can also try an
ethanol precipitation.
If your target is expressed in a sufficient amount, the best way of processing is to dilute the
cDNA sample.

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 qPCR guide

Strong background
A strong background can be due to the degradation of the probe. Check it on the NTC, and/or
on the multicomponent view, to see if the fluorescence increases without target.

A strong background can also be due to the quencher noise, as quencher such TAMRA is also
a fluorophore. Use Dark Quencher, especially for multiplex assays.

A high concentration of DNA can also lead to a strong background, if you do multiplex, try to
decrease the concentrations of individual primers and probes.

A strong background can also be explained by simple reasons such as fingerprints on the plate,
dirt in the thermocycler block, particles in the tube etc.

Genotyping troubleshooting
Probe cross hybridization or spectral overlap means that, for example, a X probe cross-
hybridizes to the Y amplicon. The following solutions can be used:

• Reduce the concentration of the “contaminating” probe, start at 300 nM and

decrease to 50 nM.
• Optimize the annealing temperature. A probe that hybridizes despite the SNP,
might not anneal at the optimal temperature. Carry out the same test by decreasing
and increasing the annealing temperature by 2 °C-steps.
• Increase the concentration of the probe that gives the lowest signal.
• Use LNA® to have a more specific X probe, for example by surrounding the SNP
by three LNA® bases.

It can also be observed that the VIC/Yakima Yellow probe signal overlaps the FAM one, or the
contrary. The same solution mentioned above can be tried. You might also try to reverse the
fluorophore keeping the same sequences, or to redesign the probes.

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 53

Frequently Asked Questions

Is a probe assay more sensitive than a SYBR® Green assay?

Both assays can be equally sensitive, however the probe assay is more specific. Consequently,
for difficult to optimize PCR assays, it is recommended to use a probe assay.

How can I get rid of a contamination from a previous PCR product?

Use a kit containing dUTP/dNTP and UNG, which will destroy previous PCR products at the
beginning of each PCR program.

Why is my Negative Control, positive?

• Simple contamination by pipetting over the NTC wells: place them as far as
possible from the lowest diluted sample (highest concentrated sample).
• The water is contaminated: use freshly purified water
• There are primer dimers in the SYBR® Green assay, leading to a late amplification.
This can be checked on the meltcurve.
• The probe is degraded.

How can I avoid gDNA being amplified in a reaction, in which I want to quantify the amount
of mRNA?

Perform a DNase treatment of the sample, or design intron spanning primers.

How can I avoid the formation of primer dimers?

Use a HotStart enzyme, optimize the design, and reduce the primer concentration.

Can I use primers I designed for classical PCR for my qPCR experiment?

Some design rules are similar, however one major change is the length of the amplicon, which
should ideally not exceed 150 bp in qPCR and can be much longer in PCR.

Why do I have several peaks on my meltcurve?

A peak before the specific peak is usually primer dimers. Two distinct melting peaks can be due
to co-amplification of genomic DNA and cDNA, or can be due to a non specific product.

What amount of template should I use in each well?

50 ng of template per well is usually enough. The maximum recommended amount of cDNA is
500 ng in 50 µl, as a too high quantity of RNA/cDNA might inhibit the qPCR reaction.

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 qPCR guide

How can I set up the threshold for my qPCR assay?

The threshold, as the baseline, is set up automatically on most machines, and calculated during
the early cycles of the qPCR run. However, you can also set it up manually.

What is an acceptable PCR efficiency for my assay?

A PCR efficiency between 96 % and 104 % is ideal. However a PCR efficiency between 90 %
and 110 % is acceptable, and the assay can usually be optimized to achieve this.

What is an acceptable R² value for my assay?

A R² higher than 0.985 is acceptable.

Should I use a kit containing ROX or not?

It depends on your machine. The ROX passive reference is mainly used on the ABI Prism® and
Eppendorf machines to normalize the signal. Check our thermocycler-kit compatibility table to
be sure you use the right kit for your machine type.

What should I give Eurogentec to get optimization / troubleshooting advice on my results?

Try to sum up the aim of your qPCR assay and the species on which you work. Then you
should send us the plate set up, the thermal cycling conditions, the kit and machine you use,
the amplification curve and associated Ct values, the standard curve, the meltcurve for SYBR®
Green assay, the raw data and multicomponent view if possible on your machine, the sequence
and Tm of your primers/probe, the concentration of primers/probe, and the normalization
method. Please send all this informations to [email protected].

How should I store kits, primers and probes?

Probe MasterMixes are stable for 2 years at -20 °C, SYBR® Green MasterMixes are stable for 1
year at -20 °C. It is also possible to store them at 4 °C for one month. Avoid doing more than 5
cycles of freeze-thawing. RT mixes are stable for one year at -20 °C or -80 °C.
Probes and primers in solution should also be stored at -20 °C in the dark, and are stable for
months.

In which buffer should probes be resuspended?

Probes are very sensitive to hydrolysis, so resuspend them in TE 0.01 or just water. Any acidic
solution will hydrolyze the probes and give a lower signal to noise ratio.

In which case is a one-step qRT-PCR recommended?

For high-throughput screening and optimized reactions, and to avoid any contamination, a
One-Step experiment is the best way to proceed.

EUROGENTEC | www.eurogentec.com | [email protected]

 55

Eurogentec products

Kits and consumables

Our MasterMixes are developed to work with standard temperature profiles, to be completely
adapted to any assay. However, it is recommended to generate a primer optimization matrix
and a primer and probe ratio matrix, as this is a crucial step to obtain the lowest signal-to-noise
ratio, the earliest Ct value and to save maximum reagents.
If you don’t find the reagent you need in the table below, please contact us at
[email protected].

EUROGENTEC | www.eurogentec.com | [email protected]

 qPCR Kits for SYBR® Assays • Compatibility with Real-Time thermocycler
ABI GeneAmp® SDS 5700 ABI Prism® 7500 iCycler iQ® Mx4000® Mx4000® Rotor-Gene™ 2000/3000/6000
ABI Prism® SDS 7000 My iQ® Mx3000P® Mx3000P® MiniOpticon®
ABI Prism® 7300/7700 iQ5 Mx3005P® Mx3005P® DNA Engine Opticon® 1 & 2
ABI Prism® 7900HT Chromo 4®
StepOne® Plus CFX96
qPCR guide

MasterCycler® ep realplex Smartcycler® 1 & 2

with ROX without ROX Quantica®
normalisation normalisation LC480

qPCR MasterMixes with ROX passive reference Reference #RXNs Sample reference
25 µl

qPCR MasterMix for SYBR® Green I (5 x 1.5 ml) RT-SN2X-03T 600 a RT-SN2X-005+

qPCR MasterMix Plus for SYBR® Green I dTTP, 7.5 ml RT-SN2X-03+WOUN 600 a (1)

®
qPCR MasterMix Plus for SYBR Green I w/o UNG, 7.5 ml RT-SN2X-03WOU+ 600 a (1)

qPCR MasterMix Plus 7,5ml for SYBR® Green I RT-SN2X-03+ 600 a RT-SN2X-005+

qPCR MasterMix Plus 15 ml for SYBR® Green I RT-SN2X-06+ 1200 a RT-SN2X-005+

qPCR MasterMix Plus 50 ml for SYBR® Green I RT-SN2X-20+ 4000 a RT-SN2X-005+

®
One step qRT-PCR MasterMix for SYBR Green I, 7.5 ml RT-SNRT-032X 600 a RT-SNRT-0052X

Two step qRT-PCR MasterMix for SYBR® Green I RT-SN2X-03RT 600 a (1)

EUROGENTEC | www.eurogentec.com | [email protected]

®
MESA GREEN qPCR MasterMix Plus for SYBR Assay dTTP, 7.5 ml RT-SY2X-03+WOUN 600 a RT-SY2X-005+WOUN

MESA GREEN qPCR MasterMix Plus for SYBR® Assay, 7.5 ml RT-SY2X-03+WOU 600 a RT-SY2X-005+WOU

®
MESA GREEN qPCR MasterMix Plus for SYBR Assay, 15 ml RT-SY2X-06+WOU 1200 a RT-SY2X-005+WOU

MESA GREEN qPCR MasterMix Plus for SYBR® Assay, 50 ml RT-SY2X-20+WOU 4000 a RT-SY2X-005+WOU

®
MESA BLUE qPCR MasterMix Plus for SYBR Assay, 7.5 ml RT-SY2X-03+WOUB 600 a RT-SY2X-005+WOUB

®
MESA FAST qPCR MasterMix Plus for SYBR Assay, 7.5 ml RT-SY2X-03+WOUF 600 a RT-SY2X-005+WOUF

One step MESA GREEN qRT-PCR MasterMix for SYBR® Assay, 7.5 ml RT-SYRT-032X 600 a RT-SYRT-0052X

(1) Use the RT-SN2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results • dTTP kit will perform slightly better that this sample kit • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample
at the recommended concentration.
 qPCR Kits for SYBR® Assays • Compatibility with Real-Time thermocycler
ABI GeneAmp® SDS 5700 ABI Prism® 7500 iCycler iQ® Mx4000® Mx4000® Rotor-Gene™ 2000/3000/6000
ABI Prism® SDS 7000 My iQ® Mx3000P® Mx3000P® MiniOpticon®
ABI Prism® 7300/7700 iQ5 Mx3005P® Mx3005P® DNA Engine Opticon® 1 & 2
ABI Prism® 7900HT Chromo 4®
StepOne® Plus CFX96
MasterCycler® ep realplex Smartcycler® 1 & 2
with ROX without ROX Quantica®
normalisation normalisation LC480

qPCR Core kits with ROX passive reference Reference #RXNs Sample reference
25 µl

qPCR Core kit for SYBR SYBR® Green I RT-SN10-05 1000 a (1)

qPCR Core kit for SYBR SYBR® Green I dTTP RT-SN10-05NU 1000 a (1)

®
Two step qRT-PCR Core kit for SYBR Green I RT-SNRT-05 1000 a (1)

qPCR MasterMixes with Low ROX passive reference

qPCR MasterMix Plus for SYBR® Green I Low ROX, 7.5 ml RT-SN2X-03+WOULR 600 a a /

®
MESA GREEN qPCR MasterMix Plus for SYBR Assay Low ROX, 7.5 ml RT-SY2X-03+WOULR 600 a a RT-SY2X-005+WOULR

MESA GREEN qPCR MasterMix Plus for SYBR® Assay Low ROX, 15 ml RT-SY2X-06+WOULR 1200 a a RT-SY2X-005+WOULR

®
MESA GREEN qPCR MasterMix Plus for SYBR Assay Low ROX, 50 ml RT-SY2X-20+WOULR 4000 a a RT-SY2X-005+WOULR

MESA BLUE qPCR MasterMix Plus for SYBR® Assay Low ROX, 7,5 ml RT-SY2X-03+WOULRB 600 a a RT-SY2X-005+WOULRB

MESA FAST qPCR MasterMix Plus for SYBR® Assay Low ROX, 7,5 ml RT-SY2X-03+WOULRF 600 a a RT-SY2X-005+WOULRF

qPCR MasterMixes without ROX passive reference

qPCR MasterMix Plus for SYBR® Green I No ROX, 7.5 ml RT-SN2X-03+NR 600 a a RT-SN2X-005+NR

®
Two step qRT-PCR MasterMix for SYBR Green I No Rox RT-SN2X-03NRRT 600 a a (2)

®
MESA GREEN qPCR MasterMix Plus for SYBR Assay No ROX, 7.5 ml RT-SY2X-03+NRWOU 600 a a RT-SY2X-005+NRWOU

MESA GREEN qPCR MasterMix Plus for SYBR® Assay No ROX, 15 ml RT-SY2X-06+NRWOU 1200 a a RT-SY2X-005+NRWOU

MESA GREEN qPCR MasterMix Plus for SYBR® Assay No ROX, 50 ml RT-SY2X-20+NRWOU 4000 a a RT-SY2X-005+NRWOU
57

(1) Use the RT-SN2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results • dTTP kit will perform slightly better that this sample kit • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample
at the recommended concentration.

EUROGENTEC | www.eurogentec.com | [email protected]

(2) Use the RT-SN2X-005+NR sample to get a valid preview of the MasterMixes and Core Kits results • dTTP kit will perform slightly better that this sample kit • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix
sample at the recommended concentration.
 qPCR Kits for SYBR® Assays • Compatibility with Real-Time thermocycler
ABI GeneAmp® SDS 5700 ABI Prism® 7500 iCycler iQ® Mx4000® Mx4000® Rotor-Gene™ 2000/3000/6000
ABI Prism® SDS 7000 My iQ® Mx3000P® Mx3000P® MiniOpticon®
ABI Prism® 7300/7700 iQ5 Mx3005P® Mx3005P® DNA Engine Opticon® 1 & 2
ABI Prism® 7900HT Chromo 4®
StepOne® Plus CFX96
qPCR guide

MasterCycler® ep realplex Smartcycler® 1 & 2

with ROX without ROX Quantica®
normalisation normalisation LC480

qPCR MasterMixes without ROX passive reference Reference #RXNs Sample reference
25 µl

MESA BLUE qPCR MasterMix Plus for SYBR® Assay No ROX, 7.5 ml RT-SY2X-03+NRWOUB 600 a a RT-SY2X-005+NRWOUB

MESA FAST qPCR MasterMix Plus for SYBR® Assay No ROX, 7.5 ml RT-SY2X-03+NRWOUF 600 a a RT-SY2X-005+NRWOUF

One step MESA GREEN qRT-PCR MasterMix for SYBR Assay No ROX, 7.5 ml RT-SYRT-032XNR 600 (a) (b) (a) a a RT-SYRT-0052XNR

qPCR Core kits without ROX passive reference

qPCR Core kit for SYBR Green I No ROX RT-SN10-05NR 1000 (a) (b) (a) a a (1)

®
Two step qRT-PCR Core kit for SYBR Green I No ROX RT-SNRT-05NR 1000 (a) (b) (a) a a (1)

qPCR MasterMixes with fluorescein

qPCR MasterMix Plus for SYBR® Green I with fluorescein, 7.5 ml RT-SN2X-03+NRFL 600 /

EUROGENTEC | www.eurogentec.com | [email protected]

®
MESA Green qPCR MasterMix Plus for SYBR Assay w/ fluorescein, 7.5 ml RT-SY2X-03+WOUFL 600 a RT-SY2X-03+WOUFL

®
MESA Green qPCR MasterMix Plus for SYBR Assay w/ fluorescein, 15 ml RT-SY2X-06+WOUFL 1200 a RT-SY2X-03+WOUFL

MESA Green qPCR MasterMix Plus for SYBR® Assay w/ fluorescein, 50 ml RT-SY2X-20+WOUFL 4000 a RT-SY2X-03+WOUFL

MESA BLUE qPCR MasterMix Plus for SYBR® Assay w/fluorescein, 7.5 ml RT-SY2X-03+WOUFLB 600 RT-SY2X-005+WOUFLB

(1) Use the RT-SN2X-005+NR sample to get a valid preview of the MasterMixes and Core Kits results. dTTP kit will perform slightly better that this sample kit. For RT reagents, combine this sample to our RT enzyme by simply adding the RT to the mastermix at the recommended concentration.
(a) In combination with ROX passive reference • RT-PARE-03
(b) In combination with fluorescein additive • RT-FLUO-ADD
 qPCR Kits for Probe Assays • Compatibility with Real-Time thermocycler
ABI GeneAmp® SDS 5700 ABI Prism® 7500 iCycler iQ® Mx4000® Mx4000® Rotor-Gene™ 2000/3000/6000
ABI Prism® SDS 7000 My iQ® Mx3000P® Mx3000P® MiniOpticon®
ABI Prism® 7300/7700 iQ5 Mx3005P® Mx3005P® DNA Engine Opticon® 1 & 2
ABI Prism® 7900HT Chromo 4®
StepOne® Plus CFX96
MasterCycler® ep realplex Smartcycler® 1 & 2
with ROX without ROX Quantica®
normalisation normalisation LC480

qPCR MasterMixes with ROX passive reference Reference #RXNs Sample reference
25 µl

qPCR MasterMix, 5 x 15 ml RT-QP2X-03 600 a RT-QP2X-005+

qPCR MasterMix Plus, 7.5 ml RT-QP2X-03-075+ 600 a RT-QP2X-005+

qPCR MasterMix Plus, 15 ml RT-QP2X-03-15+ 1200 a RT-QP2X-005+

qPCR MasterMix Plus, 50 ml RT-QP2X-03-50+ 4000 a RT-QP2X-005+

FAST qPCR MasterMix Plus, 7.5 ml RT-QP2X-03+F 600 a RT-QP2X-005+F

FAST BLUE qPCR MasterMix Plus, 7.5 ml RT-QP2X-03+FB 600 a RT-QP2X-005+FB

qPCR MasterMix Plus w/o UNG, 7.5 ml RT-QP2X-03WOU+ 600 a RT-QP2X-005+WOU

qPCR MasterMix Plus, 7.5ml QGS RT-QP2X-03+QGS 600 a RT-QP2X-005+QGS

qPCR MasterMix Plus dTTP, 7.5 ml RT-QP2X-03+WOUN 600 a RT-QP2X-005+WOUN

FAST qPCR MasterMix Plus dTTP, 7.5 ml RT-QP2X-03+WOUNF 600 a RT-QP2X-005+WOUNF

FAST BLUE qPCR MasterMix Plus dTTP, 7.5 ml RT-QP2X-03+WOUNFB 600 a RT-QP2X-005+WOUNFB

One step qRT-PCR MasterMix, 7.5 ml RT-QPRT-032X 600 a RT-QPRT-0052X

Two step qRT-PCR MasterMix RT-QP2X-03RT 600 a (1)

qPCR Core kits with ROX passive reference

qPCR Core kit RT-QP73-05 1000 a (1)

qPCR Core kit dTTP RT-QP10-05NU 1000 a (1)

(1) Use the RT-QP2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample at the recommended concentration.
59

EUROGENTEC | www.eurogentec.com | [email protected]

 qPCR Kits for Probe Assays • Compatibility with Real-Time thermocycler
ABI GeneAmp® SDS 5700 ABI Prism® 7500 iCycler iQ® Mx4000® Mx4000® Rotor-Gene™ 2000/3000/6000
ABI Prism® SDS 7000 My iQ® Mx3000P® Mx3000P® MiniOpticon®
ABI Prism® 7300/7700 iQ5 Mx3005P® Mx3005P® DNA Engine Opticon® 1 & 2
ABI Prism® 7900HT Chromo 4®
StepOne® Plus CFX96
qPCR guide

MasterCycler® ep realplex Smartcycler® 1 & 2

with ROX without ROX Quantica®
normalisation normalisation LC480

qPCR Core kits with ROX passive reference Reference #RXNs Sample reference
25 µl

qPCR Core kit only dUTP RT-QP10-05OU 1000 a (1)

Two step qRT-PCR Core kit RT-QPRT-05 1000 a (1)

qPCR MasterMixes with Low ROX passive reference

qPCR MasterMix Plus Low ROX, 7.5 ml RT-QP2X-03+WOULR 600 a a RT-QP2X-005+WOULR

FAST qPCR MasterMix Plus Low ROX, 7.5 ml RT-QP2X-03+WOULRF 600 a a RT-QP2X-005+WOULRF

FAST BLUE qPCR MasterMix Plus Low ROX, 7.5 ml RT-QP2X-03+WOULRFB 600 a a RT-QP2X-005+WOULRFB

One step qRT-PCR MasterMix Low ROX, 7.5 ml RT-QPRT-032XLR 600 a a (2)

qPCR MasterMixes without ROX passive reference

EUROGENTEC | www.eurogentec.com | [email protected]

qPCR MasterMix Plus No ROX, 7.5 ml RT-QP2X-03NR 600 a a a RT-QP2X-005+NR

FAST qPCR MasterMix Plus No ROX, 7.5 ml RT-QP2X-03+NRF 600 a a a RT-QP2X-005+NRF

FAST BLUE qPCR MasterMix Plus No ROX, 7.5 ml RT-QP2X-03+NRFB 600 a a a RT-QP2X-005+NRFB

One step qRT-PCR MasterMix No ROX, 7.5 ml RT-QPRT-032XNR 600 a a a RT-QPRT-0052XNR

Two step qRT-PCR MasterMix No ROX RT-QP2X-03NRRT 600 a a a (3)

qPCR Core kits without ROX passive reference

qPCR Core kit No ROX RT-QP73-05NR 1000 (a) a a a (3)

Two step qRT-PCR Core kit No ROX RT-QPRT-05NR 1000 (a) a a a (3)

(1) Use the RT-QP2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample at the recommended concentration.
(2) Use the RT-QPRT-0052XNR sample and add ROX passive reference to the reaction buffer at the recommended concentration (see ROX TDS).
(3) Use the RT-QP2X-005+NR sample to get a valid preview of the MasterMixes and Core Kits results • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample at the recommended concentration.
(a) In combination with ROX passive reference • RT-PARE-03
 qPCR miscellaneous
Fluorescein additive RT-FLUO-ADD 1 ml UNG (Uracil-N-Glycosylase) RT-0610-03 300 U

ROX passive reference RT-PARE-03 300 µl UNG (Uracil-N-Glycosylase) RT-0610-15 1500 U

Reverse Transcriptase Core kit RT-RTCK-03 600 RXNs (25 µl) EuroScript/RNAse inhibitor mix RT-0125-ER 3750 U

Reverse Transcriptase Core kit RT-RTCK-05 1000 RXNs (25 µl)

qPCR Internal Positive Control

Internal Positive Control Yakima Yellow ** - TAMRA RT-IPCY-T02 200 RXNs (50 µl)

Internal Positive Control Yakima Yellow ** - BHQI RT-IPCY-B02 200 RXNs (50 µl)

* For all One-Step RT kits

** Equivalent to VIC ®

qPCR validated internal controls

ß-2 Phospholipase
Target gene 18S rRNA 28S rRNA ß-actin GAPDH Ubiquitin C HMBS RPL13a SDHA HPRT1 TBP
microglobulin A2

RT-CKFT-18S
Reference RT-CKYD-28S RT-CKYD-ACTB RT-CKYD-GAPD RT-CKYD-UBC RT-CKYD-B2M RT-CKYD-YWHAZ RT-CKYD-HMBS RT-CKYD-RPL13A RT-CKYD-SDHA RT-CKYD-HPRT1 RT-CKYD-TBP
RT-CKYD-18S

Relative expression level Very high Very high High High High High Medium Medium Medium Medium Low Low

cDNA 3 3 3 3 3 3 3 3 3 3 3 3

gDNA 3 3 3 3 3 3

Organism H, Ra, M, Rb H, M H H H H H H H H H H

Amplicon length 121 bp 87 bp 91 bp 86 bp 76 bp 112 bp 93 bp 119 bp 92 bp 118 bp 100 bp 145 bp

H : human • Ra : rat • M : mouse • Rb : rabbit

CKFT : FAM-TAMRA probe
CKYD : Yakima Yellow (VIC ® equivalent) - Eclipse ® Dark Quencher probe

Each kit contains enough reagents for up to 250 - 50 µl reactions.

61

Other dye-quencher combinations are available on request.

EUROGENTEC | www.eurogentec.com | [email protected]

 96-well plate compatibility with Real-Time thermocyclers

iQ5
LC480

My iQ®
Mx4000®
Chromo 4
Quantica®

iCycler iQ®
Mx3000P®
Mx3005P®

ABI Step One

MiniOpticon®

ABI Step One Plus

ABI GeneAmp® 5700

qPCR guide

ABI Prism® SDS 7000

ABI Prism® SDS 7300
ABI Prism® SDS 7500
ABI Prism® SDS 7700
DNA Engine Opticon® 1
DNA Engine Opticon® 2

ABI Prism® SDS 7900HT

MasterCycler ep realplex

ABI Prism® SDS 7500 FAST

ABI Prism® SDS 7900 FAST
qPCR 96-well plate type Cover

Sub-skirted, high profile, frosted or white type (ABI systems) Flat caps
(RT-FLAT-300) a a a a a a
RT-PL96-AB
RT-PL96-ABW Adhesive seals
(RT-OPSL-XX**) a a a a a a

Sub-skirted, low profile, frosted or white type (ABI FAST systems)

Adhesive seals
a a a
RT-PL96-AF (RT-OPSL-XX**)
RT-PL96-AFW

Non-skirted, high profile, natural or white type Flat caps

(RT-FLAT-300) a a a a a a a a a a a a a a
RT-PL96-MQ
RT-PL96-MQW

EUROGENTEC | www.eurogentec.com | [email protected]

Adhesive seals
(RT-OPSL-XX**) a a a a a a a a a a a a a a

Non-skirted, low profile, frosted or white type Flat caps

(RT-FLAT-300) a a a a a a a
RT-PL96-OP
RT-PL96-OPW Adhesive seals
(RT-OPSL-XX**) a a a a a a

Non-skirted, low profile, white Flat caps

a
(RT-FLAT-300)
RT-PL96-LC480
Adhesive seals
(RT-OPSL-XX**) a

qPCR optical semi-dome 8-cap strips are also available (RT-OCAP-300: 300 strips). They can be used on several machines.
* plate must be cut to size (48 well) to fit Step One or MiniOpticon
** XX: RT-OPSL-25 for 25 seals / RT-OPSL-100 for 100 seals
 63

Primers and probes

Since 1987, Eurogentec has been committed to providing oligonucleotides of the highest
quality matching all research needs. Our oligonucleotide synthesis service offers custom
synthesis in large variety of scales with a wide range of modifications (FAM, DragonFly
Orange®, Yakima Yellow®, TAMRA, BHQ, Deep Dark Quencher...) and purification levels:

• Unmodified and Modified Primer Pairs

• Double-Dye Probes
• LNA® Double-Dye Probes
• Molecular Beacons
• Standard and Duplex Scorpions®
• LC Hybridization Probes
• Plexor Primer Pairs

Eurogentec know-how currently meets the satisfaction of more than 35 000 users
worldwide.

And if you became the next Eurogentec’s customer for PCR and qPCR oligonucleotides...

If you need help with the design of your oligonucleotides, information about prices
and delivery estimation or any other question, please contact our Customer HelpDesk,
staffed by qualified and experienced scientists in Molecular Biology and Chemistry at
[email protected].

EUROGENTEC | www.eurogentec.com | [email protected]

 qPCR guide

references

ABOU-BACAR A., PFAFF A.W., GEORGES S., LETSHER-BRU V., FILISETTI D., VILLARD O., ANTONI E., KLEIN J.P., CONDOLFI E., “Role of
NK cells and gamma interferon in transplacental passage of toxoplasma gondii in mouse model of primary infection”, Infection and Immunity,
Vol.72 (3), 1397-1401, 2004

AFONINA I., ANKOUDINOVA I., MILLS A., LOKHOV S., HUYNH P., MAHONEY W., “Primers with 5’ flaps improve real-time PCR”, Biotechniques,
Vol.43, 770-774, 2007.

BUSTIN S., “Absolute quantification of mRNA using Real-Time reverse transcriptin polymerase chain reaction assays”, Journal of Molecular
Endocrinology, Vol.25, p.169-193, 2000

BUSTIN S., “Quantification of mRNA using Real-Time reverse transcription PCR (RT-PCR); trends and problems”, Journal of Molecular
Endocrinology, 29, 23-39, 2002

COOK NL., VINK R., DONKIN JJ., VAN DEN HEUVEL C., “Validation of reference genes for normalization of real-time quantitative RT-PCR data
in traumatic brain injury.”, J Neurosci Res., 2008.

ERALI M., VOELKERDING KV., WITTWER CT., “High resolution melting applications for clinical laboratory medicine.”, Exp Mol Pathol.
,85(1):50-8, 2008.

FLEIGE S. and PFAFFL MW., “RNA integrity and the effect on the real-time qRT-PCR performance”, Mol Aspects Med. 27(2-3): 126-139, 2006

FREEMAN W.M., WALKER S.J., VRANA K.E., “Quantitative Rt-PCR:pitfalls and potential”, Biotechniques, Vol.112 (22), 124-125, 1999

HALFORD W.P., “The essential prerequisites for quantitative RT-PCR: pitfalls and potential”, Nature Biotechnology, Vol.17 (19), 835, 1999

HERNANDEZ M., PLA M., ESTEVE T., PRAT S., PUIDOMENECH P. and FERRANDO A., “A specific real-time quantitative PCR detection system
for event MON810 in Maize YieldGard® based on the 3’-transgene integration sequence”, Transgenic Research, Vol.12, 179-189, 2003

HOLLAND P., ABRAMSON R.D., WATSON R. and GELLAND D.H., “Detection of specific polymerase chain reaction product by utilizing the 5’-3’
exonuclease activity of thermos aquatus.”, Proc. Natl, Acad, Sci. USA, Vol.88, 7276-7280, 1991

HOOGEWIJS D., HOUTHOOFD K., MATTHIJSSENS F., VANDESOMPELE J., VANFLETEREN JR., “Selection and validation of a set of reliable
reference genes for quantitative sod gene expression analysis in C. elegans.”, BMC Mol Biol. 9:9, 2008

ILVANDERBROUCKE I., VANDESOMPELE J., DEPAEPE A. and MESSIAEN L., “Quantification of splice variants using Real-Time PCR”, Nucleic
Acids Research, Vol.29 (13), 2001

ISHII T., SOOTOME H., YAMASHITA K., “Practical evaluation of universal conditions for four-plex quantitative PCR.”, Anal Bioanal Chem.
388(1):271-8, 2007

IVANOVA A., ROSCH N., “The structure of LNA:DNA hybrids from molecular dynamics simulations: the effect of locked nucleotides.”,

J Phys Chem A.111(38):9307-19, 2007

JU J., RUAN C., FULLER C.W., GLAZER A.A. and MATHIES R.A., ‘’Fluorescence energy transfer dye-labeled primers for DNA sequencing and
Analysis’’, Proc. Natl. Acad. Sci. USA., Vol.92, 4347-4351, 1995

KARKARE S., BHATNAGAR D., “Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and
morpholino.”, Appl Microbiol Biotechnol. 71(5):575-86, 2006

KUBISTA M., ANDRADE JM., BENGTSSON M., FOROOTAN A., JONAK J., LIND K., SINDELKA R., SJOBACK R., SJOGREEN B., STROMBOM
L., STAHLBERG A., ZORIC N., “The real-time polymerase chain reaction”, Mol Aspects Med. 27(2-3):95-125, 2006.

LE CANN P., RANARIJAONA S., MONOEHO S., LEGYADER S., FERRE V., “Quantification of human astroviruses in sewage using real-time
RT-PCR”, Res. Microbiol., Vol.155 (1), 11-15, 2003

LETERTRE C., PERELLE S., DILASSE F., ARAR K., FACH P., “Evaluation of the performance of LNA and MGB probes in 5’ nuclease assays”,
Molecular and cellular probes, Vol.17 (6), 307-11, 2003

LI Y., ZHOU X., YE D., “Molecular beacons: an optimal multifunctional biological probe.”, Biochem Biophys Res Commun. 5;373(4):457-61,
2008

MASON M., PROVERO P., VAIRA A.M. and PAOLO P., ACCOTTO, “Estimating the number of integrations in transformed plants by quantitative
real-time PCR”,.BMC Biotechnology, Vol.2 (20), 2002

MYAKISHEV M., KHRIPIN Y., MU S., DEAN M., ‘’High-Throughput SNP Genotyping by Allele-Specific PCR with Universal Energy-Transfer-
Labeled primers’’, Genome Research, Vol.1, 163-169, 2001

MULLIS K. and FOBONA F., In methods in Enzymology, Vol.155, 335, 1987

OVERBERGH L., GIULIETTI A., VALCKS D., DECALLONNE B., BOUILLON R. and MATHIEU C. J., “The use of real-time transcriptase PCR for
the quantification of Cytokine Gene Expression”, Biomol.Tech, Vol.14, 33-43, 2003

PATTYN F., ROBBRECHT P., DE PAEPE F., SPELEMAN F., VANDESOMPELE J., « RTPrimersDB : the real-time PCR primer and probe database,
major update 2006”. Nucleic Acids Res 34, D684-8, 2006

EUROGENTEC | www.eurogentec.com | [email protected]

 65

SCHMITTGEN TD., LEE E.J., JIANG J., SARKAR A., YANG L., ELTON T.S., CHEN C., “Real-time PCR quantification of precursor and matire
microRNA.” Methods 44, 31-8, 2008

SCHMITTGEN TD., LIVAK KJ., “Analyzing real-time PCR data by the comparative C(T) method.” Nat Protoc. 3(6):1101-8, 2008

SCIPIONI A., MAUROY A., ZIANT D., SAEGERMAN C., THIRY E., “A SYBR Green RT-PCR assay in single tube to detect human and bovine
noroviruses and control for inhibition.”, Virol J. 5(1):94, 2008

SELLARS MJ., VUOCOLO T., LEETON LA., COMAN GJ., DEGNAN BM., PRESTON NP., “Real-time RT-PCR quantification of Kuruma shrimp
transcripts: a comparison of relative and absolute quantification procedures.”, J Biotechnol. 129(3):391-9, 2007

SOLINAS A., BROWN L.J., MXKEEN C., MELLOR J.M., NICOL J., THELWELL N., BROWN T., “Duplex Scorpions primers in SNP analysis and
FRET applications”, Nucleic Acids Res, Vol.29, E96, 2001

TAKACS T., JENEY C., KOVACS L., MOZES J., BENCZIK M., SEBE A., “Molecular beacon-based real-time PCR method for detection of 15
high-risk and 5 low-risk HPV types.”, J Virol Methods.;149(1):153-162, 2008

TYAGI S., MARRAS S.A.E. and KRAMER F.R., ‘’Wavelength-Shifting Molecular Beacons’’, Nature Biotechnology, Vol.18, 1191-1196, 2000

VANDESOMPELE J., DE PRETER K., PATTYN F., POPPE B., VAN ROT N., DE PAEPE A. and SPELEMAN F., “Accurate normalization of real time
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EUROGENTEC | www.eurogentec.com | [email protected]

 qPCR guide

Disclaimers

The polymerase Chain Reaction (PCR) process is covered by patents owned Hoffmann-La Roche, Inc. use of the PCR process requires a
license.

A license under U.S. Patents 4,683,202, 4,683,195 and 4,965,188 or their foreign counterparts, owned by Roche Molecular Systems, Inc. and F.
Hoffmann-La Roche Ltd (Roche”), has an up-front fee component and a running-royalty component. The purchase price of this product includes
limited, non-transferrable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain
Reaction (“PCR”) and related processes described in said patents solely for the research and development activities of the purchaser when this
product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights to the up-front fee component
must be obtained by the end user in order to have a complete license. These rights under the up-front fee component may be purchased from
Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler. No right to perform or offer commercial services of any kind using
PCR, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is hereby granted by
implication or estoppel. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director
of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or the Licensing Department at Roche Molecular
Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.

Use of UDG employs U.S. Patents 5,035,996, 5,945,313, 5,683,896 and their foreign counterparts licensed to Eurogentec S.A. from Invitrogen
Corporation.

Cyclic-substituted unsymmetrical cyanine dyes are covered by U.S. Patents 5,436,134 and 5,658,751 and licensed to Eurogentec S.A. by
Molecular Probes, Inc. in the direct research field.

The use of certain fluorogenic probes in 5’ nuclease assays may be covered by U.S. Patents 5,210,015 and 5,487,972, owned by Roche
Molecular Systems, Inc., and by U.S. Patent 5.538.848, owned by The Perkin-Elmer Corporation. The purchase of Eurogentec’s product does
not provide a license to use this patented technology. A license must be obtained by contacting the Director of Licensing Applied Biosystems,
850 Lincoln Centre Drive, Foster City, CA 94404 or the Licensing Department at Roche Molecular Systems Inc., 1145 Atlantic Avenue, Almeda,
CA 94501.

The use of Scorpions® Probes for research purposes is covered by a license to Eurogentec S.A. from DxS Ltd. No rights to perform or offer
diagnostic, commercial testing or other commercial services for money or money’s worth are granted by the supply of this product. Should you
wish to use this product for any other purpose not covered by this license, please contact DxS Limited at Manchester Incubator Building, 48
Grafton Street, Manchester, M13 9XX.

The use of Custom Molecular Beacon Probes employs the following patent rights licensed to Eurogentec S.A. from The Public Health Research
Institute of the City of New York: [a] the claims of U.S. Patent Application Serial No. 08/439,819; [b] the claims of U.S. Patent Application Serial
No. 08/990,176; and [c] all patent application claims and all patent claims, U.S. and foreign, that cover improvements in the foregoing claimed
probes, kits and assays and whose subjects are inventions and discoveries that are made by The Public Health Research Institute of New
York. The use of Custom Molecular Beacon Probes is limited to the field of use comprising the internal use by an end user of a product solely
in assays of the end user [or in applications of the end user’s customer, if the end user is performing contract research] in scientific research
and development.

The oligonucleotides probes and primers using the Yakima Yellow®‚ amidite and the Eclipse®‚ Non-Fluorescent Quencher are for research
purposes only, and may not be used for commercial, clinical, diagnostic or any other use. These products or portions thereof are subject to
proprietary rights of Epoch Biosciences, Inc. and are made and sold under license from Epoch Biosciences, Inc. under the patent rights WO
0142505 and other pending patent applications. There is no implied license for commercial use with respect to these products. A license must
be obtained directly from Epoch Biosciences, Inc. with respect to any proposed commercial use of these products.

LNA oligonucleotides produced under license from Exiqon® A/S for research use only. Not for resale or for therapeutic use or use in humans.

Alexa Fluor® dyes are provided under license from Molecular Probes, Inc., for research use only, and are covered by U.S. Patents 5,696,157
and 6,130,101 and their foreign counterparts.

Texas Red®, Rhodamine Red™, Marina Blue®‚ dyes are provided under license from Molecular Probes, Inc., for research use only, and are
covered by U.S. Patents 4,774,339; 5,132,432; 5,227,487; 5,274,113; 5,696,157; 5,798,276; 5,830,912; 5,846,737; 5,955,612; 6,162,931 and
their foreign counterparts.

Trademarks

ABI Prism® is a registered trademark of The Perkin-Elmer Corp. Amplifluor® is a registered trademark of Intergen Alexa®, Alexa Fluor®, Marina
Blue®, SYBR®, SYBR gold® and Texas Red® are registered trademarks of Molecular Probes, Inc. Cy® and Cy®5 Direct are registered trademarks
of Amersham Biosciences Corp. DNA Engine Opticon® is a registered trademark of MJ Research, Inc. Eclipse®, MGB®, MGB Eclipse® and
Yakima Yellow® are registered trademarks of Epoch Biosciences, Inc. Exiqon® is a registered trademark of Exiqon A/S GeneAmp® and TaqMan®
are registered trademarks of Roche Molecular Systems, Inc. GoldStar® is a registered trademark of Eurogentec S.A. HyBeacons® is a registered
trademark of LGC. I-Core® and Smartcycler® are registered trademarks of Cepheid, Inc. iCycler iQ® is a registered trademark of Bio-Rad
Laboratories, Inc. Mx3000p® and Mx4000® are registered trademarks of Stratagene Primer Express® is a registered trademark of Applera
Corporation. Oligo® Primer Analysis Software is a registered trademark of Molecular Biology Insights Inc. Quantica® is a registered trademark
of Techne, Ltd and Techne, Inc. Scorpions® is a registered trademark of DxS Ltd Uniprimer® is a registered trademark of Serologicals Royalty
Company, Rotor-Gene™ is a registered trademark of Corbett Life Science.

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 EUROPEAN OFFICES US OFFICE

BELGIUM NORTH AMERICA

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Ref.: q&qPCR booklet-0708-V2

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