real-time PCR & RT-PCR (1)
real-time
PCR & RT-PCR (2)
real-time
PCR & RT-PCR (3)
qPCR
- a
technique enabling fast,
quantitative and reliable results
Some of the limitations of
end-point
detection in (RT-) PCR
have been assuaged in real-time
PCR
systems, various are now on the market.
These systems
offer many general technical advantages,
including reduced probabilities of variability and
contamination, as
well as online monitoring and the lack
of need for postreaction analyses. Further,
some of these systems were developed with
contemporary applications
such as quantitative PCR, multiplexing,
and
high-throughput analysis in mind. In real-time
quantitative PCR
techniques, signals (generally fluorescent) are
monitored as they are generated and are tracked
after they rise above background but before the
reaction reaches
a plateau. Initial template levels can be
calculated by analyzing the shape of the curve or
by determining when
the signal rises above some threshold
value. Several commercially available real-time
PCR systems are overviewed and/or summarized in
the following sub-page.
Each of
these systems employs either one
of several general types of
fluorescent probes for detection. Several
different basic types of fluorescent probes are
used for real-time PCR
applications. Some assays employ general
dyes that bind preferentially to double-stranded
DNA (SYBR Green 1). Others use target
sequence-specific reagents such as exonuclease
probes, hybridization
probes, or molecular beacons. Although more
expensive,
sequence specific probes add specificity to the
assay, and enable multiplexing
applications. Real time PCR
or RT-PCR offers numerous advantages over previous
attempts at
quantitating (RT-)PCR. Other methods typically
rely on end-point measurements, when
often the reaction has gone beyond the exponential
phase because of
limiting reagents. To compensate for such
problems, competitive
PCR was devised,
which allows for normalization of the end
product based on the ratio between target and
competitor. Because this method is cumbersome,
requiring
a carefully constructed competitor target for each
(RT-)PCR reaction
and a series of dilutions to ensure that there
is a suitable ratio
of target to competitor, it is seldom used
successfully (absolute
quantification). In
contrast, with real time (RT-)PCR, the dynamic
range is much greater than that of competitive
(RT-)PCR - up
to 8 orders of magnitude as compared to
one
with competitive (RT-)PCR -, post-reaction
processing
is eliminated, and the measurements
are taken from the exponential range of the
reaction, where component concentrations are not
limiting. And best of
all, the entire process is automated.
qPCR, dPCR, NGS – A
journey
Jim F. Huggett, Justin O’Grady, Stephen
Bustin
Biomolecular Detection and Quantification,
available
online 15 January 2015
Scientific conferences fulfill many roles, but one
of the most
important ones is that they help shape the
direction in which a
scientific discipline grows by promoting
person-to-person exchanges of
information, ideas and constructive criticisms
between scientists from
different backgrounds. This interaction also helps
to identify areas of
controversy and promotes efforts to address and,
it is hoped, resolve
them. This year is the 30th anniversary of the
publication of the first
practical description of the polymerase chain
reaction [1], arguably
one of the simplest and the most widely used
molecular technology. It
also sees the 7th
instalment of the
Freising PCR meetings http://www.qPCR-NGS-2015.net,
which are the longest established, continuous and
most influential
conferences in this field and have provided a
looking glass for
conceptual and technical innovation as well as
practical applications
of PCR-associated methods.

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Good
Practice Guide for the Application of
Quantitative PCR (qPCR)
Author
-
Nolan T, Huggett J, Sanchez E
http://www.lgcgroup.com
qPCR
Guide
CoverThe polymerase chain reaction (PCR)
is a rapid, sensitive, and
rather simple technique to amplify DNA,
using oligonucleotide primers,
dNTPs and a heat stable Taq polymerase.
With the introduction of
real-time PCR in the late nineties, the
PCR method overcame an
important hurdle towards becoming ‘fully
quantitative’ (and therefore
known as quantitative PCR, or qPCR).
Currently, qPCR is regarded as the
‘gold standard’ in the quantitative
analysis of nucleic acids, be it
DNA, RNA or micro-RNA molecules. The
main reasons for its success are
its high sensitivity, robustness, good
reproducibility, broad dynamic
quantification range, and very
importantly, affordability.
However,
completing
qPCR assays to a high standard of
analytical quality can be
challenging for a number of reasons,
which are discussed in detail in
this guide. qPCR has a large number of
applications in a wide range of
areas, including healthcare and food
safety. It is therefore of
paramount importance that the results
obtained are reliable in
themselves and comparable across
different laboratories.
This
guide is
aimed at individuals who are starting to
use qPCR and realise that,
while this method is easy to perform in
the laboratory, numerous
factors must be considered to ensure
that the method will be applied
correctly. The guide aims to assist
those who are, or will be, using
qPCR by discussing the issues that need
consideration during
experimental design. The guide entails
“tried and tested” approaches,
and troubleshoots common issues.
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Reverse transcription quantitative PCR is an
established, simple
and effective method for RNA measurement. However,
technical
standardisation challenges combined with frequent
insufficient
experimental detail render replication of many
published findings
challenging. Consequently, without adequate
consideration of
experimental standardisation, such findings may be
sufficient for a
given publication but cannot be translated to
wider clinical
application. This article builds on earlier
standardisation work and
the MIQE guidelines, discussing processes that
need consideration for
accurate, reproducible analysis when dealing with
patient samples. By
applying considerations common to the science of
measurement
(metrology), one can maximise the impact of gene
expression studies,
increasing the likelihood of their translation to
clinical tools.

Meeting Report -
Developments in real-time PCR research and
molecular diagnostics
Stephen A Bustin
Expert Review of Molecular Diagnostics
September 2010, Vol. 10, No. 6, Pages
713-715
This meeting was designed to highlight the
wide range of
new methods,
instruments and applications that underlie the
popularity of
quantitative real-time PCR technology in all areas
of life science
research, as well as in clinical diagnostics. It
provided a fascinating
snapshot of current trends and novel approaches,
as well as important
issues concerning assay design, optimization and
quality control issues.
Quantitative
real-time RT-PCR - a perspective
Bustin
SA, Benes V, Nolan T, Pfaffl MW.
Institute
of Cellular and Molecular Science, Barts and
the London, Queen Mary's School
of
Medicine and Dentistry, University of
London, London, UK.
J
Mol Endocrinol. 2005 Jun;34(3):597-601
The
real-time
reverse transcription polymerase chain
reaction (RT-PCR) uses
fluorescent reporter molecules to monitor
the production of
amplification products during each cycle of
the PCR reaction. This
combines the nucleic acid amplification and
detection steps into one
homogeneous assay and obviates the need for
gel electrophoresis to
detect amplification products. Use of
appropriate chemistries and data
analysis eliminates the need for Southern
blotting or DNA sequencing
for amplicon identification. Its simplicity,
specificity and
sensitivity, together with its potential for
high throughput and the
ongoing introduction of new chemistries,
more reliable instrumentation
and improved protocols,
has
made real-time RT-PCR the benchmark
technology for the detection
and/or comparison of RNA levels.
The
paper has been
frequently cited by other researchers:
=> 918 times until
April 2016

Methods Vol 50 (4)
April 2010
edited
by Michael W. Pfaffl
Table
of
content:
Full
papers
and reviews
Sponsored
Application
Notes
|
The
ongoing evolution of qPCR
A summary
of interesting papers
&
reviews, presented at the qPCR 2010
Event in Vienna
The
polymerase chain reaction (PCR) is
usually described as a simple,
sensitive and rapid technique that uses
oligonucleotide primers, dNTPs
and a heat stable Taq polymerase to
amplify DNA. It was invented by
Kary B. Mullis and co-workers in the
early eighties, who were
awarded the 1993 Nobel Prize for
chemistry for this discovery. With the
discovery of real-time PCR in the
nineties the method took an important
hurdle towards becoming “fully
quantitative”. The addition of an
initial reverse-transcription (RT) step
produced the complementary
RT-PCR, a powerful means of amplifying
any type of RNA. Today
quantitative PCR (qPCR) is widely used
in research and diagnostics,
with numerous scientists contributing to
the pre-eminence of PCR in a
huge range of DNA-, RNA- (coding and
non-coding) or protein- (immuno-
or proximity ligation assay qPCR) based
applications. Soon the PCR was
regarded as the “gold standard” in the
quantitative analysis of nucleic
acid, because of its high sensitivity,
good reproducibility, broad
dynamic quantification range, easy use
and reasonable good value for
money.
qPCR has substantial
advantages in
quantifying low target copy numbers
from limited amounts of tissue or
identifying minor changes in mRNA or
microRNA expression levels in samples
with low RNA concentrations or
from single cells analysis. The
extensive potential to quantify
nucleic acids in any kind of biological
matrix has kept qPCR at the
forefront of extensive research efforts
aimed at developing new or
improved applications. But are qPCR and
its associated quantification
workflow really as simple as we assume?
It is essential to have a
comprehensive
understanding of the underlying
basic principles, error sources and
general problems inherent with qPCR
and RT-qPCR. This rapidly reveals the
urgent need to promote efforts
towards more reproducible, sensitive,
truly quantitative and,
ultimately, more biologically valid
experimental approaches. Therefore,
the challenge is to develop assays that
meet current analytical
requirements and anticipate new
problems, for example in novel
biological matrices or for higher
throughput applications.
Unfortunately, we are far from having
developed optimal workflows, the
highest sensitivity, the best RNA
integrity metrics or the ultimate
real-time cycler, all of which are
indispensable for optimal PCR
amplification and authentic results. The
qPCR research community still
aims to improve and evolve, which brings
to the topic of this PCR
special issue - The ongoing evolution of
qPCR.
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Methods Vol 59(1)
January 2013
edited
by Michael W. Pfaffl
Table
of
content
Full
papers
and
reviews
Sponsored
Application
Notes
|
Transcriptional
Biomarkers
A
summary of
interesting papers &
reviews, presented at the qPCR & NGS
2013 Event in
Freising-Weihenstephan
Biological
markers
(biomarkers) have been used for
diagnostic testing for more
than 50 years and have acquired
immense scientific and clinical
value.
This process has accelerated in
the 21st century, leading to
their
growing appeal as markers for
routine diagnostic practice.
There are
numerous promising biomarkers,
the most important of which are
currently used for assessing the
efficacy of treatment,
development of
new drugs, especially in the
area of therapeutic medicine for
cancer or
cardiovascular diseases. In the
past, biomarkers were defined as
‘cellular, biochemical or
molecular alterations that are
measurable in
biological media such as human
tissues, cells, or body fluids’.
Nowadays the term biomarker is
defined as ‘a characteristic
that is
objectively measured and
evaluated as an indicator of
normal biological
processes, pathogenic processes,
or pharmacologic responses to a
therapeutic intervention or
other health care intervention’
by the
Biomarker Consortium of the
Foundation for the National
Institutes of
Health (FNIH). A biomarker
should be able to reveal a
specific
biological trait or a measurable
change in the organism, which is
directly associated with a
physiological condition or
disease status.
Early
disease
detection by biomarkers offers
an effective opportunity for
enhancing disease detection,
improving patient prognosis and
streamlining the use of drug
therapy and assessing clinical
outcomes of
treatment. Hence biomarkers are
potentially useful along several
steps
of the disease process:
- Before
diagnosis,
they provide the potential for
screening and risk assessment.
- As part
of the
diagnostic process, biomarkers
can determine staging,
grading, and
selection of initial therapy.
- Subsequently,
in the
treatment phase, they can be
used to monitor therapy
success, select
additional therapies or
monitor recurrent diseases.
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Real-Time
PCR -- Understanding Ct
Real-time PCR, also called quantitative
PCR or qPCR, can provide a
simple and elegant method for determining
the amount of a target
sequence or gene that is present in a
sample. Its very simplicity can
sometimes lead to problems of overlooking
some of the critical factors
that make it work. This review will
highlight these factors that must
be considered when setting up and
evaluating a real-time PCR reaction.
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The Quantitative
PCR
Technical Guide from Sigma-Aldrich is
intended to provide new
users with an introduction to qPCR, an
understanding of available
chemistries, and the ability to apply qPCR
to answer research
questions. The guide also contains numerous
tips and tools for the
experienced qPCR user.
PCR
Technologies Guide
qPCR and MIQE
Seminar Series
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Real-time
PCR is a form of polymerase chain
reaction (PCR) in which
data are
collected in real-time as the reaction
proceeds. Continuous data
collection enables one of the principal
applications of real-time PCR,
target quantitation. Because quantitation
is among the most common uses
for real-time PCR, it is often referred to
as quantitative PCR or qPCR.
Life
Technologies™
offers tools to provide reliable real-time
results the
first time and every time. With trusted
Applied Biosystems®
instruments
and software, TaqMan® Assays and master
mixes tailored for success,
and
innovative products for new real-time PCR
research applications, such
as digital PCR, castPCR™ rare sequence
detection, and even products for
protein analysis, we can accelerate your
real-time PCR research.
Real-time
PCR guide - Theory of real-time
PCR
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PrimeTime®
qPCR Application Guide
Experimental Overview, Protocol, and
Troubleshooting. The qPCR
Application Guide is intended to provide
guidance to users on the
entire qPCR process, from RNA isolation to
data analysis. Click to
download a pdf of the PrimeTime qPCR
Application Guide.
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Download our
new PCR and RT-PCR technical
brochure
Demanding applications such as long-range
and multiplex PCR present
challenges for scientists. Download our new
qualitative PCR and RT-PCR
brochure to find out how to achieve the best
results from your PCR
methods.
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Real-time PCR
Application guide
By Bio-Rad
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EUROGENTEC
BOOKLETS
This
brochures are very appriated one, on which
we had lots of positive
reactions in the sense of:
- finally
a company, who can give me a full overview
- finally a
company, who is not preoccupied by a
certain system
- great ! it
makes me better understand real time PCR
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Quantification
of mRNA using real-time
RT-PCR
Tania Nolan,
Rebecca
E Hands & Stephen A Bustin
Nature Protocols
(2006) Vol. 1, No. 3; p1559-1582

The real-time
reverse
transcription polymerase chain reaction (RT-qPCR)
addresses the evident
requirement for quantitative data analysis in
molecular medicine, biotechnology, microbiology and
diagnostics and has
become the method of choice for the quantification
of
mRNA. Although it is often described as a ‘‘gold’’
standard, it is far
from being a standard assay. The significant
problems caused by
variability of RNA templates, assay designs and
protocols, as well as
inappropriate data normalization and inconsistent
data
analysis, are widely known but also widely
disregarded. As a first step
towards standardization, we describe a series of
RT-qPCR
protocols that illustrate the essential technical
steps required to
generate quantitative data that are reliable and
reproducible. We
would like to emphasize, however, that RT-qPCR data
constitute only a
snapshot of information regarding the quantity of a
given transcript in a cell or tissue. Any assessment
of the biological
consequences of variable mRNA levels must include
additional information regarding regulatory RNAs,
protein levels and
protein activity. The entire protocol described
here, encompassing
all stages from initial assay design to reliable
qPCR data analysis,
requires approximately 15 h.

The
real-time polymerase chain reaction
Kubista M, Andrade JM, Bengtsson M,
Forootan
A, Jonak J, Lind K, Sindelka R, Sjoback R,
Sjogreen B, Strombom L,
Stahlberg A, Zoric N.
Mol Aspects Med. 2006
27(2-3): 95-125.
TATAA Biocenter, Medicinargatan 7B, 405 30
Goteborg,
Sweden
The scientific,
medical, and diagnostic communities have been
presented the most
powerful tool for quantitative nucleic acids
analysis: real-time PCR
[Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL
Press, San Diego,
CA]. This new technique is a refinement of the
original Polymerase
Chain Reaction (PCR) developed by Kary Mullis and
coworkers in the mid
80:ies [Saiki, R.K., et al., 1985. Enzymatic
amplification of
beta-globin genomic sequences and restriction site
analysis for
diagnosis of sickle cell anemia, Science 230,
1350], for which Kary
Mullis was awarded the 1993 year's Nobel prize in
Chemistry. By PCR
essentially any nucleic acid sequence present in a
complex sample can
be amplified in a cyclic process to generate a
large number of
identical copies that can readily be analyzed.
This made it possible,
for example, to manipulate DNA for cloning
purposes, genetic
engineering, and sequencing. But as an analytical
technique the
original PCR method had some serious limitations.
By first amplifying
the DNA sequence and then analyzing the product,
quantification was
exceedingly difficult since the PCR gave rise to
essentially the same
amount of product independently of the initial
amount of DNA template
molecules that were present. This limitation was
resolved in 1992 by
the development of real-time PCR by Higuchi et al.
[Higuchi, R.,
Dollinger, G., Walsh, P.S., Griffith, R., 1992.
Simultaneous
amplification and detection of specific
DNA-sequences. Bio-Technology
10(4), 413-417]. In real-time PCR the amount of
product formed is
monitored during the course of the reaction by
monitoring the
fluorescence of dyes or probes introduced into the
reaction that is
proportional to the amount of product formed, and
the number of
amplification cycles required to obtain a
particular amount of DNA
molecules is registered. Assuming a certain
amplification efficiency,
which typically is close to a doubling of the
number of molecules per
amplification cycle, it is possible to calculate
the number of DNA
molecules of the amplified sequence that were
initially present in the
sample. With the highly efficient detection
chemistries, sensitive
instrumentation, and optimized assays that are
available today the
number of DNA molecules of a particular sequence
in a complex sample
can be determined with unprecedented accuracy and
sensitivity
sufficient to detect a single molecule. Typical
uses of real-time PCR
include pathogen detection, gene expression
analysis, single nucleotide
polymorphism (SNP) analysis, analysis of
chromosome aberrations, and
most recently also protein detection by real-time
immuno PCR.
CHAPTER
7 - Quantitative Real-time PCR Analysis
JACQUIE T. KEER
The
sensitivity
of analysis achievable with PCR has led to the
technology being adopted across a range of
sectors. For many
applications a quantitative result is required,
which has driven the
development of a range of strategies to deter¬mine
the amount of
starting material in a sample. Approaches such as
com¬petitive PCR1
and limiting dilution analysis2 have been used as
routes to
quantification, although the variable nature of
the PCR process and the
amplification of the target to a maximal level
irrespective of the
starting amount of target limit the accuracy of
these methods. The
advent of kinetic or real-time PCR4 has overcome
many of the
limita¬tions of earlier strategies, by monitoring
the increase in
product generated during the course of the
reaction, in ‘real time’.
Quantitative approaches are based on the time or
cycle at which
amplification is first detected, rather than
requiring quantification
of PCR products, and the principle is illustrated
schematically in
Figure 7.1. A range of samples of known target
content are usually
amplified together with the samples under test,
and the accumulation of
PCR product in each cycle is determined.
Alternatively the signal from
two targets may be compared to determine a
relative measure of
quantification, and this is often used in
measurement of gene
expression which is considered in more detail in
Chapter 9.
Here a fluorescent reporter assay is used to
monitor increase in
fluorescence at each PCR cycle. The point at which
the signal becomes
detectable, or crosses some arbitrary threshold
value, is determined
for each standard and sample. These values are
then plotted against the
amount of target in the standards to produce a
calibration curve, and
the amount of target in the unknown samples can
then be interpolated
from the graph. The linear relationship between
the amount of starting
material and the
measured cycle threshold (Ct) values are
maintained across several
orders of magnitude, so assays based on
quantitative PCR (qPCR) have an
unusually large dynamic range. There are a number
of other significant
benefits in using real-time PCR analysis,
including the greatly
increased sensitivity associated with the use of
fluorescent reporters
and signal collection devices, and the rapid
cycling times that are
achievable on some instruments. In addition,
homo¬geneous qPCR
assays minimise the potential for
cross-contamination com¬pared
with conventional methods as reaction vessels need
not be opened in
order to analyse amplification products, and also
avoid variation
introduced by gel analysis. In short, real-time
PCR offers the
potential of well-characterised and
highly sensitive quantitative analysis, although
the diversity of
instruments, detection chemistries, data handling
methods and the lack
of quantitative reference standards present
significant challenges to
measurement comparability.........
The
MIQE
Guidelines - Minimum
Information for
Publication of
Quantitative Real-Time PCR Experiments
Stephen
A.
Bustin,
Vladimir Benes, Jeremy A. Garson,
Jan Hellemans, Jim
Huggett, Mikael
Kubista, Reinhold Mueller, Tania
Nolan, Michael W.
Pfaffl, Gregory L. Shipley Jo
Vandesompele, and Carl T.
Wittwer
Clinical
Chemistry
2009, 55(4): 611-622
BACKGROUND:
Currently,
a lack of consensus exists on how best to
perform and interpret quantitative real-time PCR
(qPCR) experiments.
The problem is exacerbated by a lack of sufficient
experimental detail in many publications, which
impedes a
reader's ability to evaluate critically the
quality of the
results presented or to repeat the experiments.
CONTENT:
The
Minimum Information for Publication of
Quantitative
Real-Time PCR Experiments (MIQE) guidelines target
the
reliability of results to help ensure the
integrity of the
scientific literature, promote consistency between
laboratories, and increase experimental
transparency. MIQE
is a set of guidelines that describe the minimum
information necessary
for evaluating qPCR experiments. Included is a
checklist to
accompany the initial submission of a manuscript
to the publisher. By
providing all relevant experimental conditions and
assay
characteristics, reviewers can assess the validity
of the
protocols used. Full disclosure of all reagents,
sequences,
and analysis methods is necessary to enable other
investigators to reproduce results. MIQE details
should be
published either in abbreviated form or as an
online
supplement.
SUMMARY:
Following
these
guidelines will encourage better experimental
practice,
allowing more reliable and unequivocal
interpretation of
qPCR results.
Go the MIQE
sub-domain
Real-Time PCR:
Current Technology and Applications
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
http://www.horizonpress.com/realtimepcr
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
The
Road from Qualitative to Quantitative
Assay. What is next?
by Michael W. Pfaffl
Chapter 8
in "The PCR
Revolution" edited by Stephen A.
Bustin, page 110 - 128
Cambridge University Press
The PCR reaction is widely used in many
applications throughout the
world. It has it secure place in the molecular
biological history as
one of the most revolutionary methods ever. The
principles of PCR are
clear, but how the reaction procedure can be
optimized and how to bring
out the best? Where are the fields of
improvements? What is
the status quo and what is next?
"The PCR
Revolution" edited by Stephen A.
Bustin - book cover -
table of content
SPUD
-
a quantitative PCR assay for the detection of
inhibitors in
nucleic
acid preparations.
Nolan
T, Hands RE, Ogunkolade W, Bustin SA.
Anal
Biochem.
2006 351(2): 308-310

Among the many
factors that determine the sensitivity, accuracy,
and
reliability of a real-time quantitative reverse
transcription
polymerase chain reaction (qRT–PCR)1 assay, template
quality is one of
the most important determinants of reproducibility
and biological
relevance [1]. This is a well-recognized problem
[2], and there are
numerous reports that describe the significant
reduction in the
sensitivity and kinetics of qPCR assays caused by
inhibitory components
frequently found in biological samples [3], [4],
[5], [6], [7] and [8].
The inhibiting agents may be reagents used during
nucleic acid
extraction or copurified components from the
biological sample such as
bile salts, urea, haeme, heparin, and immunoglobulin
G. At best,
inhibitors can generate inaccurate quantitative
results; at worst, a
high degree of inhibition will create false-negative
results. The most
common procedure used to account for any differences
in PCR
efficiencies between samples is to amplify a
reference gene in parallel
with the reporter gene and to relate their
expression levels. However,
this approach assumes that the two assays are
inhibited to the same
degree. The problem is even more pronounced in
absolute quantification,
where an external calibration curve is used to
calculate the number of
transcripts in the test samples, an approach that is
commonly adopted
for quantification of pathogens. Some, or all, of
the biological
samples may contain inhibitors that are not present
in the nucleic acid
samples used to construct the calibration curve,
leading to an
underestimation of the mRNA levels in the test
samples [9]. The
increasing interest in extracting nucleic acids from
formalin-fixed
paraffin-embedded (FFPE) archival material
undoubtedly will lead to an
exacerbation of this problem. Obviously, such
inhibitors are likely to
distort any comparative quantitative data. However,
a recent survey of
practices revealed that only 6% of researchers test
their nucleic acid
samples for the presence of inhibitors
[10]..............
PCR
technology is
based on a
simple principle; an enzymatic reaction that
increases the initial
amount of nucleic acids. This method makes it
possible to detect
specific mRNA transcripts in any biological
sample. Performing RT-PCR
analysis does not only comprehend this
experimental PCR step. Following
the whole workflow of a RT-PCR quantitative
analysis, it starts with
the sampling step, followed by nucleic acid
extraction and
stabilization, cDNA synthesis and finally the qPCR
where the mRNA
quantification takes place. Problems arise when
optimization of the
experimental work flow becomes necessary because
of high technical
variations. The PCR reaction itself is a quite
stable reaction with
reproducibility between 2-8%. Therefore the source
of experimental
variances can often be found in the pre-PCR
analytical steps. Usually
this is neglected and optimization is done for PCR
reaction only. In
this chapter – RT-PCR optimization strategies -
the whole workflow of
RT-PCR experiment will be discussed, because the
identification of the
source of variability is only possible following
error accumulation in
every single step. Reliable data can be created
when the technical
variance caused by the experimental steps is kept
as low as possible.
In this chapter many recommendations to decrease
the technical variance
can be found.
REVIEW:
RNA integrity and the
effect on the real-time qRT-PCR performance.
Fleige
S
& Pfaffl MW.
Mol
Aspects Med. 2006 27(2-3): 126-139
The
assessment of
RNA
integrity is a critical first step in obtaining
meaningful gene
expression data. Working with
low-quality RNA may strongly compromise the
experimental results of
downstream
applications which are often labour-intensive,
time-consuming, and
highly expensive.
Using intact RNA is a key element for the successful
application of
modern molecular biological methods,
like qRT-PCR or micro-array analysis. To verify RNA
quality nowadays
commercially
available automated capillary-electrophoresis
systems are available
which are
on the way to become the standard in RNA quality
assessment. Profiles
generated yield information on RNA
concentration, allow a visual inspection of RNA
integrity, and generate
approximated
ratios between the mass of ribosomal sub-units. In
this
review, the importance of RNA quality for
the qRT-PCR was analyzed by determining the RNA
quality of different
bovine tissues and
cell culture. Independent analysis systems are
described and compared
(OD measurement,
NanoDrop, Bioanalyzer 2100 and Experion). Advantage
and disadvantages
of RNA
quantity and quality assessment are shown in
performed applications of
various tissues and cell cultures.
Further the comparison and correlation between the
total RNA integrity
on PCR
performance as well as on PCR efficiency is described.
On the basis of
the derived results we can argue that
qRT-PCR performance is affected by the RNA integrity
and PCR efficiency in
general is not
affected by the RNA integrity. We
can recommend a RIN higher than five as good total
RNA quality and
higher than eight as perfect total RNA for
downstream application.
Go the RNA Integrity
sub-domain
Quantitative
real-time PCR for cancer detection: the lymphoma
case.
Stahlberg
A, Zoric N, Aman P, Kubista M.
Expert
Rev Mol Diagn. 2005 5(2): 221-230.
TATAA
Biocenter, Medicinaregatan 7B, 413 90 Gothenburg,
Sweden.

Advances
in the biologic sciences
and technology are providing molecular targets for
diagnosis and
treatment of
cancer. Lymphoma is a group of cancers with diverse
clinical courses.
Gene
profiling opens new possibilities to classify the
disease into subtypes
and guide a
differentiated treatment. Real-time PCR is
characterized by high
sensitivity,
excellent precision and large dynamic range, and has
become the method
of
choice for
quantitative gene expression measurements. For
accurate gene
expression profiling by real-time PCR, several
parameters must be
considered and
carefully validated. These include the use of
reference genes and
compensation
for PCR inhibition in data normalization.
Quantification by real-time
PCR
may be
performed as either absolute measurements using an
external standard,
or as
relative measurements, comparing the expression of a
reporter gene with
that of a presumed constantly expressed reference
gene. Sometimes it is
possible to compare expression of reporter genes
only, which improves
the accuracy
of prediction. The amount of biologic material
required for real-time
PCR
analysis is
much lower than that required for analysis by
traditional methods
due to
the very high sensitivity of PCR. Fine-needle
aspirates and even
single cells contain enough material for accurate
real-time PCR
analysis.
Real-time
PCR for
mRNA quantitation
Marisa L. Wong and Juan F. Medrano
Biotechniques 39 (2005)
Real-time
PCR has become one of the most widely used methods
of gene quantitation
because it has a large dynamic range, boasts
tremendous sensitivity,
can be highly sequence-specific, has little to no
post-amplification
processing, and is amenable to increasing sample
throughput. However,
optimal benefit from these advantages requires a
clear understanding
of the many options available for running a
real-time PCR experiment.
Starting with the theory behind real-time PCR,
this review discusses
the key components of a real-time PCR experiment,
including one-step
or two-step PCR, absolute versus relative
quantitation, mathematical
mod-els available for relative quantitation and
amplification
efficiency
calculations, types of normalization or data
correction, and detection
chemistries. In addition, the many causes of
variation as well as
methods to calculate intra- and inter-assay
variation are addressed.
Comment
and response on
Wong
and Medrano’s
“Real-time PCR
for
mRNA quantification”
BioTechniques
39: 75-85 (July 2005)
Martin Dufva
Technical
University
of Denmark, Lyngby, Denmark

Absolute
quantification of mRNA using real-time
reverse
transcription PCR assays.
Bustin
SA
Journal of Molecular Endocrinology 25: 169-193 (
2000)
The
reverse transcription polymerase chain reaction
(RT-PCR) is the
most sensitive method for the detection of
low-abundance
mRNA, often obtained from limited tissue samples.
However, it is a complex
technique, there are substantial problems associated
with its true
sensitivity, reproducibility and
specificity
and, as a quantitative method, it suffers
from the problems inherent in PCR. The recentintroduction
of
fluorescence-based kinetic RT-PCR procedures
significantly simplifies
the process of producing reproducible quantification
of mRNAs and
promises to overcome these limitations. Nevertheless,
their successful
application depends on a clear understanding of the
practical problems, and
careful
experimental design, application and validation
remain
essential for accurate quantitative measurements
of transcription.
This review discusses the technical aspects
involved, contrasts conventional
and
kinetic RT-PCR methods for quantitating gene
expression and compares the different
kinetic
RT-PCR systems. It illustrates the usefulness of
these assays
by demonstrating the significantly
different
levels of transcription between
individuals of the housekeeping gene family,
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
Quantification
of mRNA using real-time reverse transcription
PCR: trends and problems.
Bustin
SA. J Mol Endocrinol. 2002 29:
23-29 Review
The
fluorescence-based
real-time reverse transcription PCR
(RT-PCR) is widely used for the quantification
of steady-state
mRNA levels and is a critical tool for basic
research, molecular medicine and biotechnology.
Assays are easy to perform, capable of
high throughput, and can combine high sensitivity
with reliable
specificity. The technology is evolving rapidly
with the introduction
of new enzymes,
chemistries and instrumentation. However, while
real-time RT-PCR
addresses many of the difficulties
inherent in
conventional RT-PCR, it has become
increasingly clear that it engenders new problems
that require urgent
attention. Therefore, in addition to providing a
snapshot of the state-of-the-art
in
real-time RT-PCR, this review has an additional
aim: it
will describe and discuss critically
some of
the problems associated with interpreting
results that are numerical and lend themselves
to statistical
analysis, yet whose accuracy is significantly
affected by
reagent and operator variability.
Validities of mRNA
quantification using recombinant RNA and
recombinant DNA external
calibration curves in real-time RT-PCR
M. W. Pfaffl
& M. Hageleit
Biotechnology Letters (2001)
23, 275-282
Reverse
transcription (RT) followed by polymerase chain
reaction
(PCR) is the technique of choice for analysing mRNA
in extremely low
abundance. Real-time RT-PCR using SYBR Green I
detection combines the
ease and necessary exactness to be able to produce
reliable as well as
rapid results. To obtain high accuracy and
reliability in RT and
real-time PCR a highly defined calibration curve is
needed.
We have developed, optimised and validated an
Insulin-like growth
factor-1 (IGF-1) RT-PCR in the LightCycler, based on
either a
recombinant IGF-1 RNA (recRNA) or a recombinant
IGF-1 DNA (recDNA)
calibration curve. Above that, the limits, accuracy
and variation of
these externally standardised quantification systems
were determined
and compared with a native RT-PCR from liver total
RNA. For the
evaluation and optimisation of cDNA synthesis rate
of recRNA several
RNA backgrounds
were tested. We conclude that external
calibration curve using recDNA is a better model for
the quantification
of mRNA than the recRNA calibration model. This
model showed
higher sensitivity, exhibit a larger quantification
range, had a higher
reproducibility, and is more stable than the recRNA
calibration curve.
METHODS
& REVIEWS
Quantitative Real-Time
Polymerase Chain Reaction for
the Core Facility
Using TaqMan and the Perkin-Elmer/Applied
Biosystems Division 7700
Sequence Detector
by
Deborah S.
Grove
Nucleic Acid
Facility,
Life Science Consortium, The
Pennsylvania State University, University Park, PA
16802
The
real-time TaqMan PCR
and applications in veterinary medicine
by
Christian M.
Leutenegger
REAL-TIME
PCR
by M.Tevfik
Dorak, MD, PhD
http://dorakmt.tripod.com/genetics/realtime.html
Quantitative
real-time
RT-PCR
A very short
course
Gregor L. Shipey (The University of Texas,
Houston)
Assay
Development on TaqMan System
Assay Setup
and Data Analysis
Advantage
of a high
temperature fluorescence
acquisition
during
amplification
Development and
validation of an externally standardised
quantitative Insulin like
growth factor-1
(IGF-1) RT-PCR using LightCycler SYBR ® Green I
technology.
Pfaffl, MW
(2001)
In: Meuer, S,
Wittwer, C, Nakagawara, K, eds. Rapid Cycle
Real-time PCR, Methods and
Applications
Springer Press,
Heidelberg, ISBN 3-540-66736-9

How
to Reduce Primer Dimers in a LightCycler PCR
Technical
Note No.
LC 1/1999

4th segment quantification
The
4th segment
during the amplification program melts unspecific
LightCycler PCR products
at 85°C,
eliminates the non-specific fluorescence signal
and ensures
accurate quantification
of the desired
IGF-1 products (figure 2).
High temperature
quantification keeps
the fluorescence of
the no template control around 1
unit, while the specific IGF-1 signal
rises up to 40-50
fluorescence units. SYBR ® Green
I determination at 85°C
results in reliable
and sensitive IGF-1 quantification
with high linearity (correlation
coefficient r
= 0.99) over seven orders of
magnitude (102
to 109 RNA start molecules; lower
figure). In contrast, a
conventional determination
at 72°C results in a
truncated
quantification range (r = 0.99) over only four orders
of magnitude (105
to 109 RNA start molecules; upper
figure).

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