Chapter 1 An Introduction to Real-Time PCR
N. A. Saunders
The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the real-time PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. Since the first practical demonstration of the concept real-time PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, these real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.

Chapter 2 An Overview of PCR Platforms
J. M. J. Logan and K. J. Edwards
Real-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a wide choice of instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software. The main features of each instrument are compared and factors important to weigh up when deciding on a platform are highlighted.

Chapter 3 Homogeneous Fluorescent Chemistries for Real-Time PCR
M. A. Lee, D. J. Squirrell, D. L. Leslie and T. Brown
The development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of nucleic acid quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR allowing kinetic measurement of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that use the phenomenon of Fluorescent Energy Transfer. In this chapter we describe these methods in detail, outline the principles of each process, and describe published examples. This text has been written to provide an impartial overview of the utility of different assays and to show how they may be used on various commercially available thermal cyclers.

Chapter 4 Reference Gene Validation Software for Improved Normalization
J. Vandesompele, M. Kubista and M. W. Pfaffl
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
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.

Chapter 6 Performing Real-time PCR
K.J. Edwards and J.M.J. Logan
Optimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be affected by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence. The analysis of the real-time PCR results is also an important consideration and this differs from the analysis of conventional block-based thermal cycling. Real-time PCR provides information on the cycle at which amplification occurs and on some platforms the melting temperature of the amplicon or probe can be determined.

Chapter 7 Internal and External Controls for Reagent Validation
M. A. Lee, D. L. Leslie and D. J. Squirrell
False negatives in PCR can occur from inhibition of one or more of the reaction components by a range of factors. Therefore applications requiring a high level of confidence in the result need to be designed to control for the occurrence of false negatives. While an external, or batch, control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Here we discuss the application and development of molecular mimics for use as controls in real-time PCR, and explain a number of concepts and experimental considerations that will aid in the optimisation of controlled multiplexed assays.

Chapter 8 Introduction to the Applications of Real-Time PCR
N.A. Saunders
The technique of real-time PCR has features that make its use advantageous in a wide range of applications. A number of examples, covering the main areas of application, are given in the following chapters of this book. In this introduction the important features of these applications are discussed.

Chapter 9 Analysis of mRNA Expression by Real-Time PCR
Stephen A. Bustin and Tania Nolan
Its conceptual and practical simplicity, capacity for high throughput, and combination of high sensitivity with exacting specificity has made the fluorescence-based real-time reverse transcription polymerase chain reaction (qRT-PCR or RT-qPCR) today's method of choice for the quantification of RNA. The technology continues to evolve rapidly with the introduction of new protocols, enzymes, chemistries and instrumentation and has become the "Gold Standard" for a huge range of applications in basic research, molecular medicine, and biotechnology. Progress is increasingly associated with an increased appreciation of the limitations associated with this technology and the need for careful experimental design, application and validation.

Chapter 10 Validation of Array Data
Elisa Wurmbach
Microarray techniques allow the parallel assessment of the relative expression of thousands of transcripts in response to different experimental conditions or in different tissues. The ability to correctly identify differentially expressed genes is limited by the signal to noise ratio, the variation in the levels of gene expression, and/or the variability in the measurements due to the assay itself. Therefore, an unequivocal identification of differentially expressed transcripts requires independent confirmation. Quantitative real-time RT-PCR (qPCR) is the method of choice because of its broad range of linearity. Furthermore, it can be easily adapted to systematically study tens to hundreds of different transcripts. The cDNA microarray technique is introduced as an example, followed by comparisons to different microarray platforms and their characteristics. Data analysis of microarray experiments will show the importance of verification of results. General differences between microarray hybridisations and PCR reactions and, in particular, the performance of different platforms are described and compared. Furthermore, the effects of increasing tissue complexity on detection of differentially expressed transcripts are elucidated with specific examples.

Chapter 11 Mutation Detection by Real-Time PCR
Elaine Lyon, Rong Mao and Jeffrey Swensen
Real-time applications for mutation detection include detecting alterations associated with inherited disease, acquired alterations in oncology, and microbial or viral mutations associated with drug resistance in infectious diseases. Probe chemistries described for these applications include hydrolysis (TaqMan ) and hybridisation probes (FRET and Molecular Beacons). Hydrolysis probes detect mutations by allele specific hybridisation at a specific temperature, while hybridisation probes allow dynamic detection through a temperature range. Primer chemistries are described for allele specific amplification and Scorpion primers. Recent progress in scanning amplicons for mutations also includes high resolution melting. The design of each of these methods is described, along with applications.

Chapter 12 Real-Time NASBA
Julie D. Fox, Catherine Moore and Diana Westmoreland
NASBA is an isothermal nucleic acid amplification method which is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. First developed NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been reported and applied to a wide range of RNA and some DNA targets. Thus real-time NASBA has proved to be the basis of sensitive and specific assays for detection, quantification and differentiation of RNA and DNA targets. Molecular beacons have most often been utilised in real-time NASBA whether in commercially-available kits or as published in-house developed assays. As experience in design of molecular-beacon probes increases and fluorimeters suitable for real-time NASBA become widely available this methodology will be confirmed as a suitable alternative to real-time RT-PCR (and perhaps DNA PCR).

Chapter 13 Applications in Clinical Microbiology
Andrew David Sails
The introduction of real-time PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. There has been an explosion of interest in this technique since its introduction and several hundred reports have been published describing applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents. However, its greatest impact is probably its use for the quantitation of target organisms in samples. The ability to monitor the PCR reaction in real-time allows accurate quantitation of target sequence over at least six orders of magnitude. The closed-tube format which removes the need for post-amplification manipulation of the PCR products also reduces the likelihood of amplicon carryover to subsequent reactions reducing the risk of false-positives. As more laboratories begin to utilise these methods standardisation of assay protocols for use in diagnostic clinical microbiology is needed, plus participation in external quality control schemes is required to ensure quality of testing.

Chapter 14 Diagnosis of Invasive Fungal Infections
D.S. Perlin
The mounting prevalence of invasive fungal disease in immunocompromised patients is exacerbated by inadequate methods for pathogen detection. PCR-based amplification approaches have been developed to address this problem because conventional methods for pathogen identification lack sensitivity, specificity and speed, and some infectious organisms are difficult to culture. PCR amplification of ribosomal genes and their internal transcribed spacer regions coupled with sequence-specific detection probes are the most reliable approaches for fungal identification. Real-time self-reporting probes capable of single nucleotide allelic discrimination have expanded PCR applications to target mechanisms of drug resistance. Clinical applications of PCR are expanding for diagnosing invasive fungal diseases in blood and respiratory specimens at an early stage to improve treatment outcomes for high risk patients.

Chapter 15 Biodefense
Christina Egan, Nick M. Cirino and Kimberlee A. Musser
With the public's reawakened concern regarding use of biological agents as weapons, the rapid detection, discrimination, and identification of pathogenic organisms and toxins has become a priority for state and federal government agencies. High confidence, cost effective, and near real-time diagnostic methods are essential to protecting national health security whether the target is public health, agriculture, commodities, or water supply infrastructures. While culture-based methods have been, and will likely remain, the gold standard for microbiological diagnostics, PCR-based tests offer significant advantages in sensitivity, specificity, speed and data richness that make them invaluable to diagnostic laboratories. In this chapter, we will describe the application of real-time PCR methods in biodefense. We will discuss the use of real-time PCR in biodefense in terms of general workflow and processing considerations, clinical diagnostic applications, environmental diagnostic applications, and multiplex screening. Real-time PCR assays can be either quantitative (qPCR) or qualitative, depending on whether a standard curve is included with the analytical run. Most diagnostic and biodefense applications utilise the qualitative nature of real-time PCR as a detection platform; this chapter will focus on the benefits of these types of assays. Finally, we will consider the future uses and anticipated advances in real-time PCR applications as related to biodefense.

Chapter 16 Real-Time PCR: Application to Food Authenticity and Legislation
Gordon Wiseman
Real-time PCR is now an accepted analytical tool within the food industry. Its principal role has been one of assisting the legislative authorities, major manufacturers and retailers to confirm the authenticity of foods. The most obvious role is the detection and quantification of GMOs, but real-time PCR makes a significant contribution to many other areas of the food industry, including food safety and other speciality analyses such as the detection of common wheat adulteration in pasta and the detection of allergenic species. The role of quantitative real-time PCR in determining the actual amount of these materials, which are subject to considerable regulation, is discussed together with a consideration of the uncertainty of the methods.

Chapter 17 Molecular Haplotyping by Real-time PCR
Genevieve Pont-Kingdon, Alison Millson and Elaine Lyon
Molecular real-time PCR methods can determine whether two or more mutations are on the same or different chromosomes. This ability to haplotype without family studies is useful for research and clinical purposes and can give an advantage over genotyping. Haplotyping by real-time PCR with hybridization probes has been demonstrated for adjacent repeats and single base alterations, with a probe that covers both sites. However, base alterations may be separated by distances greater than a traditional hybridisation probe will cover. We described a probe design that covers both (or all) sites, but does not include the entire sequence between the sites. When hybridized with the template, the template is forced to form a loop. This "loop-out" probe will dissociate from the template as a unit, therefore allowing haplotyping of base alterations separated by over 80 bp. Examples of haplotyping by traditional probes for adjacent sequence variants, as well as examples of "loop-out" probes are presented