Relative
Quantification
or
Comparative Quantification
in qPCR
Relative
quantification determines the changes in
steady-state mRNA levels of
a gene across multiple samples and expresses it
relative to the levels
of an internal control RNA. This reference gene is
often a housekeeping
gene and can be co-amplified in the same tube in a
multiplex assay or
can
be amplified in a separate tube. Therefore, relative
quantification
does
not require standards with known concentrations and
the reference can
be
any transcript, as long as its sequence is known.
Relative
quantification
is based on the expression levels of a target gene
versus one or more
reference
gene(s)
and in many experiments it is adequate for
investigating physiological
changes
in gene expression levels. To calculate the
expression of a target gene
in relation to an adequate reference gene various
mathematical models
are
established. Calculations are based on the
comparison of the distinct
cycle
determined by various methods, e.g. crossing points
(CP) and cycle
threshold
values
(Ct) at a constant level of fluorescence; or CP
acquisition according
to
established mathematic algorithm. To date
several calculation
mathematical models have
been
developed
calculating the relative expression ratio.
A new
mathematical
model
for relative quantification
in
real-time RT-PCR
Pfaffl
Michael W.
Nucleic
Acids Res. 2001 29(9):
E45
| Use of the real-time
polymerase chain reaction (PCR) to amplify
cDNA products reverse
transcribed from mRNA is on the way to
becoming a routine tool in
molecular biology to study low abundance gene
expression. Real-time PCR
is easy to perform, provides the necessary
accuracy and produces
reliable as well as rapid quantification
results. But accurate
quantification of nucleic acids requires a
reproducible methodology and
an adequate mathematical model for data
analysis. This study enters
into the particular topics of the relative
quantification in real-time
RT–PCR of a target gene transcript in
comparison to a reference gene
transcript. Therefore, a new mathematical
model is presented. The
relative expression ratio is calculated only
from the real-time PCR
efficiencies and the crossing point deviation
of an unknown sample
versus a control. This model needs no
calibration curve. Control levels
were included in the model to standardise each
reaction run with
respect to RNA integrity, sample loading and
inter-PCR variations. High
accuracy and reproducibility (<2.5%
variation) were reached in
LightCycler PCR using the established
mathematical model. |
|
Download
paper PDF version
|
Absolute
and
relative real-time PCR in the quantification of tst
gene expression
among methicillin-resistant Staphylococcus aureus:
evaluation by two
mathematical models.
Chini V, Foka
A,
Dimitracopoulos G, Spiliopoulou I.
Lett
Appl Microbiol. 2007 Nov;45(5):479-84.
Department
of Microbiology, School of Medicine, University of
Patras, Patras,
Greece.
AIM: Absolute
and relative quantitative real-time reverse
transcriptase polymerase
chain reaction (RT-PCR) by the use of two
mathematical models were
applied in order to study the expression of tst
gene encoding the toxic
shock syndrome toxin-1 (TSST-1), among
methicillin-resistant
Staphylococcus aureus (MRSA).
METHODS
AND
RESULTS: Thirteen epidemic MRSA belonging to
different clones and
carrying a variety of toxin genes were selected.
tst gene expression
was achieved by using absolute and relative
quantitative real-time
RT-PCR and the SYBR Green I. Absolute RT-PCR
showed a statistically
significant higher level of tst expression
among strains isolated
from
soft tissue infections. Relative quantification
was performed in
relation to 23S rRNA expression by the application
of two mathematical
models, the 2(-DeltaDeltaCt) and the Pfaffl
analysis methods.
CONCLUSIONS:
tst gene expression was best calculated by the
relative real-time
RT-PCR analysis applying the Pfaffl analysis
method, taking into
account the reactions' efficiencies. Level of tst
expression was
related to patients' infection and did not depend
on the MRSA genetic
profile.
SIGNIFICANCE
AND
IMPACT OF THE STUDY: The results indicate that the
application of the
Pfaffl analysis method in the evaluation of
relative real-time RT-PCR
is more adequate.
Real-Time PCR:
Current Technology and Applications
http://www.horizonpress.com/realtimepcr
Publisher: Caister Academic Press
Editor: Julie Logan, Kirstin Edwards and Nick
Saunders Applied and
Functional Genomics, Health Protection Agency,
London (2009)
ISBN: 978-1-904455-39-4
Chapter 4 -
Reference Gene Validation Software for
Improved Normalization
J. Vandesompele, M. Kubista and M. W.
Pfaffl (2009)
Real-time PCR is the method of choice for
expression analysis of a
limited number of genes. The measured gene
expression variation between
subjects is the sum of the true biological
variation and several
confounding factors resulting in non-specific
variation. The purpose of
normalization is to remove the non-biological
variation as much as
possible. Several normalization strategies have
been proposed, but the
use of one or more reference genes is currently
the preferred way of
normalization. While these reference genes
constitute the best possible
normalizers, a major problem is that these genes
have no constant
expression under all experimental conditions. The
experimenter
therefore needs to carefully assess whether a
certain reference gene is
stably expressed in the experimental system under
study. This is not
trivial and represents a circular problem.
Fortunately, several
algorithms and freely available software have been
developed to address
this problem. This chapter aims to provide an
overview of the different
concepts.
Chapter 5 - Data
Analysis Software
M. W. Pfaffl, J. Vandesompele and M.
Kubista (2009)
Quantitative real-time RT-PCR (qRT-PCR) is widely
and increasingly used
in any kind of mRNA quantification, because of its
high sensitivity,
good reproducibility and wide dynamic
quantification range. While
qRT-PCR has a tremendous potential for analytical
and quantitative
applications, a comprehensive understanding of its
underlying
principles is important. Beside the classical
RT-PCR parameters, e.g.
primer design, RNA quality, RT and polymerase
performances, the
fidelity of the quantification process is highly
dependent on a valid
data analysis. This review will cover all aspects
of data acquisition
(trueness, reproducibility, and robustness),
potentials in data
modification and will focus particularly on
relative quantification
methods. Furthermore useful bioinformatical,
biostatical as well as
multi-dimensional expression software tools will
be presented.
Real-Time
PCR:
Current Technology and Applications -
Book reviews:
"... a comprehensive
overview of the RT-PCR technology, which is as
up-to-date as a book can
be ..." Mareike Viebahn in Current
Issues
in Molecular Biology (2009)
"... a useful
book
for students ..." from J.
Microbiological Methods
"provides a
dual
focus by aiming, in
the early chapters, to provide both the theory
and practicalities of
this diverse and superficially simple
technology, counter-balancing
this in the later chapters with real-world
applications, covering
infectious diseases, biodefence, molecular
haplotyping and food
standards." from Microbiology
Today
"a
reference work
that should be found both in university
libraries and on the shelves of
experienced applications specialists." from
Microbiology Today
"a
comprehensive
guide to real-time PCR technology and its
applications" from Food
Science and Technology Abstracts
(2009) Volume 41 Number 6
"This
volume
should be of utmost
interest to all investigators interested and
involved in using RT-PCR
... the RT-PCR protocols covered in this book
will be of interest to
most, if not all, investigators engaged in
research that uses this
important technique ... a well balanced book
covering the many
potential uses of real-time PCR ... valuable for
all those interested
in RT-PCR." from Doodys
reviews (2009)
"provide
the
novice and the experienced user with guidance on
the technology, its
instrumentation, and its applications" from
SciTech Book News June 2009
p. 64
"...
written
by international authors
expert in specific technical principles and
applications ... a useful
compendium of basic and advanced applications
for laboratory
scientists. It is an ideal introductory textbook
and will serve as a
practical handbook in laboratories where the
technology is employed."
from Christopher J. McIver, Microbiology
Department,
Prince of Wales Hospital, New South Wales,
Australia writing in
Australian J. Med. Sci. 2009. 30(2): 59-60
Relative
Expression
Software Tool
(REST©) for group wise comparison
and statistical analysis of relative
expression results in real-time
PCR.
Michael W.
Pfaffl,
Graham W. Horgan & Leo Dempfle (2002)
Nucleic Acids
Research 2002 30(9):
E36
Summary
Use
of
the real-time polymerase chain reaction (PCR) to
amplify cDNA products
reverse transcribed
from mRNA (RT) is on the way to become a routine
tool
used in molecular biology to
study low abundant
gene expression. Real-time PCR is easy
to perform, provides the necessary
exactness
and produces reliable
as well as rapid quantification results. But accurate
quantification of
nucleic acids requires a reproducible methodology
and an adequate
mathematical model for
data analysis. This study enters into the
particular topics of the
relative quantification
in real-time RT-PCR of a target gene transcript in
comparison to a reference
gene transcript.
Therefore a new mathematical model is presented. The
relative expression ratio
is
calculated only from the real-time PCR
efficiencies and the
crossing point deviation of
an unknown sample versus a control. This model
needs no calibration
curve. Control
levels were included in the model to standardise
each reaction run with
respect to
RNA integrity,
sample loading and inter-PCR variations. High
accuracy and
reproducibility (<2.5% variation) were reached
in LightCycler PCRusing the
established
mathematical model.
Introduction
The
reverse transcription (RT) followed by polymerase
chain reaction (PCR)
is the technique of choice
to analyse mRNA
expression derived from various
sources. Real-time RT-PCR is
high sensitive and
allow a quantification of rare
transcripts and small changes in gene
expression. Beside this
it is easy to perform, provides the necessary
exactness and produces
reliable as well
as rapid quantification results. The simplest
detection technique of
newly synthesized
PCR products in real-time PCR uses SYBR Green I fluorescence
dye, that bind
specifically to the minor groove double-stranded
DNA. The quantification
method of
choice depends on the target sequence, the
expected range of the mRNA
amount present in the tissue, the degree of accuracy
required,
and
whether quantification needs to be relative or
absolute.
Generally
two
quantification types in real-time RT-PCR
are possible:
|
(A)
|
A relative
quantification based on the relative
expression of a target gene
versus
a reference gene. To
investigate the physiological changes in gene
expression,
the relative expression ratio
is adequate for the most purposes.
|
|
(B)
|
An absolute
quantification, based either on an
internally or an externally
calibration curve.
link => "absolute"
quantification |
Using
such a
calibration curve, the methododology has to be
highly validated and the identical
LightCycler
PCR amplificationefficiencies for standard
material and
target cDNA
must be confirmed. Nevertheless is the generation
of stable and reliable
standard material,
either recombinant DNA or recombinant RNA, very
time consuming and it must
be
precisely quantified. Further a normalisation of
the target gene with an
endogenous
standard is recommended. Therefore mainly non regulated
reference genes or
house keeping genes like glyceraldehyde-3-phosphate
dehydrogenase
(G3PDH
or GAPDH), albumin, actins, tubulins, cyclophilin,
18S rRNA or 28S
rRNA were applicable. House keeping genes are
present in all nucleated
cell types since they are necessary for basis cell
survival. The mRNA
synthesis of these
genes is considered to be stable and secure in various
tissues, even under
experimental treatments. But numerous studies have
already shown that the
mentioned house keeping genes are regulated and vary
under experimental
conditions. To circumvent the high expenditure of
design and production of
standard
material, as well as optimisation and validation
of a calibration curve
based
quantification model, and finally the needed normalisation
of the target
transcripts to an endogenous housekeeping
transcript, an reliable
and accurate
relative quantification model in real-time (RT-)
PCR is needed. This study
enters into the
particular topics of the relative quantification
of a target gene in
comparison to a
reference gene. A new and simple mathematical
model for data analysis
was
established, the application of the new model was
tested and compared with
available
mathematical calculation models. Derived
reproducibility, based on
intra- and inter test
variation of this relative quantification, and
accuracy of the model will
be discussed.
Real-time PCR
amplification efficiencies and linearity
Real-time
PCR efficiencies were calculated from the given
slopes in LightCycler
software. The
corresponding real-time PCR efficiency (E) of one
cycle in the
exponential phase was
calculated according to
the
equation:
E = 10 ^[–1/slope]
(Figure 1)
Link =>
alternative efficiency calculation
methods and algorithm
Investigated
transcripts showed high real-time PCR efficiency
rates for TyrA (E = 2.09),
PyrB (E =
2.16)
and Gst (E = 1.99) in the investigated range from
0.40 pg to 50 ng cDNA
input
(n = 3) with high linearity (Pearson cor. coef.
r>0.95).
Intra-assay and inter-assay
variation
To
confirm precision and reproducibility of real-time
PCR the intra-assay
precision was determined
in 3 repeats within
one LightCycler run.
Inter-assay variation was investigated
in 3
different experiment runs performed on 3
days using 3 different premix
cups of
LightCycler – Fast Start DNA Master SYBR
Green I kit (Roche
Diagnostics). Determination of variation was done
in 20 ng reverse transcribed
total RNA (Table
1).
Test reproducibility for all investigated
transcripts was low in
inter-test
experiment (<3.91%)
and even lower in intra-test experiment (<2.16%).
The calculation of
test precision and test variability is based on
the CP variation from the
CP mean
value.
Mathematical model for
relative quantification in real-time PCR
A new
mathematical model was presented to determine the
relative
quantification of a target
gene in comparison
to a reference gene. The
relative expression ratio ( R ) of a target
gene is computed
based on E and the CP deviation of a unknown
sample versus a control (Equation
1), and expressed in comparison to a
reference gene.
This
reference gene could be a stable and secure
unregulated transcript,
e.g. a house keeping
gene transcript. For the calculation of R, the
individual real-time PCR efficiencies
(E)
and the CD deviation (delta CP) of the
investigated transcripts must
be known. Real-time PCR
efficiencies were calculated, according to E =
10 [–1/slope] as shown
in Figure 1 [Etarget = real-time PCR
efficiency of target
gene transcript; Eref
=
real-time PCR efficiency of reference gene
transcript]. CP deviations of control
cDNA minus sample of
the target gene and reference genes were
calculated
according to the
derived CP values [delta CPtarget = CP
of control - CP of
sample;delta
CPref = CP of control - sample]. Mean
CP, variation of CP
and delta CP values
between control and sample of investigated
transcripts are listed in
Table 2.
Beside this, the influence of differing cDNA input
concentrations on
delta CP are shown. Intended
cDNA input
concentration variation of control and
sample were compared at
different levels (low
level: 3.2 ng, 4.0 ng, 4.8 ng cDNA;
high level: 16
ng, 20 ng and 24 ng cDNA). They resulted in stable
and
constant delta CP cycle
numbers. In Table 3 the
correspondent ratios ( R ) of
target genes in comparison
to the reference
gene were calculated, through to
the established mathematical
model (Equation
1). The expression
ratios of target genes remain
stable,
even under intended –20% or +20% cDNA variation
and low and high cDNA
input levels,
performed in two runs. A minimal coefficient of
variation (CV) of 2.50% and
1.74% was observed, respectively.
Discussion
Reverse
transcription followed by PCR is the most powerful
tool to amplify
small amounts
of mRNA. Because of its high ramping rates,
limited annealing and
elongation time, the rapid
cycle PCR in the LightCycler system offers stringent
reaction conditions
to
all PCR components and leads to a primer sensitive
and template specific
PCR. The application of fluorescence techniques to
real-time PCR combines the
PCR amplification, product detection and quantification
of newly
synthesised DNA, as well as the verification in
the melting curve
analysis. This
led
to the development of new kinetic RT-PCR methodologies
that are
revolutionising the possibilities of mRNA
quantification.
We
focused in this paper on the relative
quantification of target gene
transcripts in
comparison to a reference gene transcript. A new
mathematical model for
data analysis
was presented to calculate the relative expression
ratio on the basis of the
PCR efficiency and
crossing point deviation of the investigated
transcripts (Equation
1). The concept of the threshold fluorescence is
the
basis of an accurate
and reproducible quantification using fluorescence
based RT-PCR methodologies.
Threshold
fluorescence is defined as the point at which the
fluorescence rises
appreciably
above the background fluorescence. In the used
“Fit Point Method” the
threshold fluorescence and therefore the DNA amount
in the capillaries is
identical for all samples. A
linear
relationship between the
CP, crossing the
threshold
fluorescence, and the log of the start molecules
input
in the reaction is given.
Therefore quantification will always occur during
exponential phase, and it
will
be not affected by any reaction components becoming
limited in the plateau
phase. In the established model the relative
expression ratio of an
target gene is normalized with the expression of
an endogenous desirable
unregulated reference gene transcript to compensate
inter PCR variations
between the runs. Is the CP of the chosen
reference gene equal in
the control as well as in the sample (delta CP
= 0), stable and
constant reference gene mRNA levels are given. Under
this considerations of an
unregulated reference gene transcript no normalisation
is needed and Equation
1 can be shortened to Equation 2 and 3.
Two other
mathematical models are available for the relative
quantification during
real-time PCR. The “Efficiency
calibrated mathematical method for the relative
expression ratio
in real-time PCR” is presented by
Roche Diagnostics in a
truncated form in an
internal publication. The complete equation is in
principle the same and
results in
identical relative expression ratio like our model
(Equation 4).
But,
the way
of
calculation in the described mathematical model is
hard to understand. The
second model available, the “delta-delta Ct
method”
for comparing relative expression
results
between
treatments in real-time PCR (Equation 4) is
presented by PE Applied
Biosystems
(Perkin
Elmer, Forster City, CA, USA). The model presumes
an optimal and
identical real-time amplification efficiencies of
target and reference gene
of E =
2. “Delta-delta Ct method” is only
applicable for a quick
estimation of the
relative expression ratio. For such an quick
estimation also equation 1
can be
shortened and
transferred in Equation 4,
under the
condition that
Etarget
= Eref =
2
Our presented
formula 1 combines both models in order to better
understanding
the mode of CP
data analysis and for a more reliable and
exact relative gene expression.
Relative
quantification
is always based on an reference transcript.
Normalisation of the target
gene with an
endogenous standard was done via the
reference gene expression,
to compensate
inter-PCR variations. Beside this
further control
levels were
included in the mathematical model to standardise
each reaction run with respect
to RNA
integrity, RT efficiency or cDNA sample
loading variations.The
reproducibility of the RT
step varies greatly between
tissues, the applied RT
isolation methodology
(25) and the used RT enzymes (26).
Different cDNA inputconcentrations
were tested
on low and high cDNA input
ranges, to mimic different RT efficiencies
(± 20%) at
different quantification
levels. In the applied two-step RT-PCR, using
random hexamer primers,
all possible interferences during RT will
influence all target
transcripts as well as
the internal reference transcript in parallel.
Occurring background
interferences
retrieved from extracted tissues components, like
enzyme inhibitors, and cDNA
synthesis efficiency were related to target and reference
similarly. All
products underwent identical reaction conditions
during RT and variations
only
disappear during
real-time PCR. Any source of error during RT
will be compensated through
the model itself. Widely distributed single-step RT-PCR
models are not
applicable, because in each reaction set-up and
for each investigated
factor individual
and slightly different RT conditions will occur. Therefore
the variation in a
two-step RT-PCR will always be lower and the reproducibility
of the assay
will be higher, that in a single-step RT-PCR (8).
Reproducibility of the
developed mathematical model was dependent on
the exact determination of
real-time amplification efficiencies and on the given
low LightCycler CP
variability. In our mathematical model the needed
reliability and
reproducibility was given,
which was confirmed by high accuracy and a
relative error of <2.5%
using low and
high template concentration input.
Conclusion
LightCycler
real-time
PCR using SYBR Green I fluorescence dye is a rapid
and
sensitive method
to detect low amounts of mRNA molecules and
therefore offers important physiological
insights on mRNA
expression level. The established mathematical model
is presented in order for
a better understanding the mode of analysis in
relative
quantification in
real-time RT-PCR. It is only dependent on DCP and
amplification efficiency of
the
transcripts. No additional artificial nucleic
acids, like recombinant
nucleic acid
constructs in external calibration curve models,
are needed.
Reproducibility of
LightCycler RT-PCR in general and the minimal
error rate of the model
allows for an
accurate determination of the relative expression
ratio. Even different cDNA
input
resulted in minor variations. Relative expression
is adequate for the most
relevant
physiological expression changes. In future it is
not necessary to establish
more
complex and time consuming quantification models based
on calibration curves.
For
the differential display of mRNA the relative
expression ratio is an
ideal and simple
tool for the verification of RNA or DNA array chip
technology results.
=>
Relative Expression Software Tool (
REST © ) <=
=>
download REST
© <=
Analysis of relative
gene expression
data using real-time quantitative PCR and
the
2^[ -delta delta
Ct ] method.
Livak KJ
& Schmittgen TD. Methods 2001
Dec;25(4): 402-408
Applied
Biosystems, Foster City, California 94404, USA.
The
two most
commonly used methods to analyze data from
real-time, quantitative PCR
experiments are absolute
quantification and relative quantification.
Absolute
quantification
determines the input copy number, usually by
relating the PCR signal to
a standard
curve. Relative quantification relates the PCR
signal
of the target transcript
in
a treatment group to that of another sample
such
as an untreated control.
The 2(-delta delta Ct) method is a convenient
way
to analyze the relative
changes in gene expression from real-time quantitative
PCR experiments.
The purpose of this report is to present the
derivation,
assumptions, and
applications of the 2(-delta delta Ct) method. In
addition, we present the
derivation and applications of two variations of
the 2(-delta delta Ct)
method that
may be useful in the analysis of real-time,
quantitative
PCR data.
A
quantitative real-time
PCR method for detection of B-Lymphocyte
Monoclonality
by comparison of kappa and lambda
Immunoglobulin
Light Chain Expression
Anders
Stahlberg,1 Pierre Aman, Börje Ridell, Petter
Mostad, and Mikael Kubista
Clinical
Chemistry
49(1): 51-59
Background: An
abnormal IgL
kappa / IgLlambda ratio
has long been used as a clinical criterion for
Hodgkin
B-cell lymphomas. As a first step towards a
quantitative real-time PCR
based multi marker
diagnostic analysis of
lymphomas, we have developed a method for
determination of IgL kappa /
IgLlambda
ratio in clinical samples.
Methods: Light-up
probe
based real-time PCR was used
to quantify
,IgL kappa and IgLlambda cDNA from 20
clinical samples. The samples were also
investigated by routine immunohistochemical
analysis
and flow cytometry analysis.
Results: The
classification
of patient samples by
Q-PCR
correlated well with the routine diagnostic
data.
To account for sample and template specific PCR
inhibition in the
biological samples,
we developed a method for in
situ calibration to determine sample specific
Q-PCR efficiencies of the reactions being
compared. This increased
considerably the accuracy of the Q-PCR
method. We also designed an approach to classify
patient samples based
on average PCR
efficiencies. This allowed faster
and
more cost efficient classification suitable for
a first gross classification in high
throughput screens.
Conclusions:
This
work is a first step towards
analyzing clinical
samples using quantitative
light-up probe based
real-time PCR. Our results shows that Q-PCR
based methods are
highly suitable for high
throughput screens of suspected tumor samples by
determining
anomalous gene expression that are
characteristic
for tumor cells.
Real-Time
PCR
Technology for Cancer
Diagnostics
Philip S. Bernard and Carl T. Wittwer
Clinical Chemistry 48: 8 1178–1185 (2002)
Background:
Advances in the biological sciences and technology
are providing
molecular targets for diagnosing and treating
cancer. Current
classifications in surgical pathology for staging
malignancies are
based primarily on anatomic features (e.g.,
tumor-nodemetastasis) and
histopathology (e.g., grade). Microarrays together
with clustering
algorithms are revealing a molecular diversity
among cancers that
promises to form a new taxonomy with prognostic
and, more importantly,
therapeutic significance. The challenge for
pathology will be the
development and implementation of these molecular
classifications for
routine clinical practice. in the
clinical laboratory.
Quantitative real-time PCR can determine gene
duplications or
deletions. Furthermore, melting curve analysis
immediately after PCR
can identify small mutations, down to single base
changes. These
techniques are becoming easier and faster and can
be multiplexed.
Real-time PCR methods are a favorable option for
the analysis of cancer
markers.
Approach:
This article
discusses the benefits, challenges, and
possibilities for solid-tumor profiling in the
clinical laboratory with
an emphasis on DNA-based PCR techniques. Content:
Molecular markers can
be used to provide accurate prognosis and to
predict response,
resistance, or toxicity to therapy. The diversity
of genomic
alterations involved in malignancy necessitates a
variety of assays for
complete tumor profiling. Some new molecular
classifications of tumors
are based on gene expression,
requiring a paradigm shift in specimen processing
to preserve the
integrity of RNA for analysis. More stable markers
(i.e., DNA and
protein)
are readily handled.
Summary:
There is a
need to translate recent discoveries in oncology
research into clinical practice. This requires
objective, robust, and
cost-effective molecular techniques for clinical
trials and,
eventually, routine use. Real-time PCR has
attractive features for
tumor profiling in the clinical laboratory.
|
