real-time PCR & RT-PCR (1)
real-time PCR & RT-PCR (2)
real-time PCR & RT-PCR (3)

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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.

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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:  => 279 times until August 2010

• http://Scholar.Google.com/

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author: Prof Stephen Bustin

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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• Nice internet page from Denmark explaing real-time PCR
• Quantitative real-time PCR on WIKIPEDIA  (English)
• Quantitative real-time PCR on WIKIPEDIA  (German)
• NEW  -  Papers from HorizonPress 2008
  
• Interesting and outstanding qPCR reviews and publications
• Nature Protocols:  Quantification of mRNA using real-time RT-PCR
  
• UPDATE  => Influence of RNA integrity on real-time RT-PCR results
• Gene Expression gateway  (by Bio-Rad)
• Download our new PCR and RT-PCR technical brochure  (by Qiagen)
  
• Interesting papers dealing with single-cell qRT-PCR
  
• Interesting and outstanding qPCR publications  (see below)
• qPCR overview presentation by M. Tevfik Dorak (PDF)
• Real-time PCR by M.Tevfik Dorak (HTML)
• Real-time PCR handbook  (HTML)
  
• New tools and technologies for real-time qPCR
  
• Interesting WWW  links
• ABI - Essentials of Real Time PCR  (PDF) 
• Estimation and Determination of real-time PCR efficiency
• Real-time PCR Tutorial - University of South Carolina
• Real-time PCR using SYBR Green I  (PDF)
• real-time PCR Application Guide (by Bio-Rad 2006)
  
• Literature:  RT-PCR & real-time RT-PCR (all)
• Literature:  RT-PCR and real-time RT-PCR (in general)

	The Web Guide of PCR Technique
	Quantitative PCR: How does it work? 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.
			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.
			EUROGENTEC  BOOKLETS New Eurogentec booklet older RT-PCR booklet troubleshooting guide 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

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.

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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.

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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

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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?

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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]..............

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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.

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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.

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