real-time PCR & RT-PCR (1)
real-time PCR & RT-PCR (2)
real-time PCR & RT-PCR (3)
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.
author:
Prof 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 Web Guide of PCR
Technique
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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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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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EUROGENTEC BOOKLETS
Very
good qPCR overview ( 68 pages and 32 pages )
eurogentec-RT-PCR-booklet.pdf
eurogentec-qq-troubleshooting.pdf
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. mikael.kubista@tataa.com
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.
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
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.
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.
A
comparison of various
calibration curves (rec
RNA & rec DNA standard curves &
biological samples) using
qRT-PCR
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
"Real-Time PCR"
or Kinetic PCR
by University of Iowa DNA
Facility
A. Real-Time Chemistry
B.
Instrumentation
C.
real-Time PCR Quantitation
D.
Primer and Probe Design
E.
Thermal Cycling Parameters
F.
Sample Preparation
G.
Data and Analysis
H.
Supplemental Information
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).
Bio-Rad
Gene Expression Gateway
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
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