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.
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: => 433 times until
November 2011
edited
in 2010 by
Michael W. Pfaffl
Table
of
content:
Full
papers
and reviews
Sponsored
Application Notes
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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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author:
Stephen Bustin
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Real-time
Polymerase
Chain reaction
The real-time
polymerase chain reaction uses fluorescent reporter dyes to combine DNA
amplification and detection steps in a single tube format. The increase
in fluorescent signal recorded during the assay is proportional to the
amount of DNA synthesised during each amplification cycle. Individual
reactions are characterised by the cycle fraction at which fluorescence
first rises above a defined background fluorescence, a
parameter known as the threshold cycle (Ct) or crossing point (Cp).
Consequently, the lower the Ct, the more abundant the initial
target. This correlation permits accurate quantification of target
molecules over a wide dynamic range, while retaining the sensitivity
and specificity of conventional end-point PCR assays. The homogeneous
format eliminates the need for post-amplification manipulation and
significantly reduces hands-on time and the risk of contamination.
Real-time PCR is often abbreviated to qPCR, although that abbreviation
is not universally accepted.
There are three
main chemistries in general use:
1. Intercalating dyes, such as
SYBR-Green, which fluoresce upon light excitation when bound to double
stranded DNA. These are cheap, easily added to legacy assays and
amplification products can be verified by the use of melt curves. They
can lack specificity and fluorescence varies with amplicon length. In
general, they are one Ct or so more sensitive than probe-based assays.
2. Fluorophores attached to primers,
e.g. Invitrogen's Lux or Promega's Plexor primers. These are relatively
inexpensive and amplification products can be verified by melt curves.
Specificity depends on the primers and specific, usually
company-specific design software needs to be used for optimal
performance. This is not necessarily a bad thing (indeed the Plexor
software is very useful), but it is not always possible to change
primer design parameters.
3. Hybridisation-probe based methods,
e.g. hydrolysis (TaqMan) or Molecular Beacons. These are the most
specific, as products are only detected if the probes hybridise to the
appropriate amplification products. There are many variations on this
theme, with melt curve analysis possible for some chemistries. Their
main disadvantages are cost, complexity and occasional fragility of
probe synthesis. There are potential problems associated with the fact
that probe-based assays do not report primer dimers that can interfere
with the efficiency of the amplification 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.
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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.
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Invitrogen
The new real-time PCR guide focuses on the
theory of real-time PCR,
and covers in-depth sections on assay design, data analysis and
troubleshooting.
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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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