Quantitative real-time PCR. Methods and protocols (Q2250274)

The book under review consists of 17 chapters on the ``gold standard technology'' used for the quantification of nucleic acids. The applications range from microbiological detections to pathological applications, e.g. leukemia transplancental metastasis and kidney transplant. These examples are discussed here. The book also includes details about the importance of purity and integrity of RNA and the minimum information necessary for qRT-PCR experiments. The book commences with a brief introductory description of qPCR, highlighting the types of qPCR commonly used in practice and a selective overview of its numerous uses. In the second chapter, the authors present and discuss MIQE (minimum information for the publication of qRT-PCR) guidelines that were established in 2009 to promote a strict set of pre-assay conditions, experiment design and analysis and to avoid the publication of inconsistent, inaccurate or wrong data. This chapter also includes a plea for accuracy in the context of a constantly increasing complexity of gene pathways. The third chapter focuses on the selection of reliable reference genes though a pilot study that identifies the best candidates for a given experiment (for example, using geNorm). Issues like the stability of reference genes and the optimal number of reference genes are discussed in detail. The chapter also includes an alternative normalization method (global mean normalization) which could be appropriate for screening studies of large sets of genes, e.g. the miRNAome. The fourth chapter discusses digital PCR (dPCR) and includes a brief history of the protocol, a review of its applications as well as a set of rules for this type of experiment. In addition, the chapter includes numerical details of how to approximate the dynamic range of the dPCR reaction and on analysis of the enrichment effect. The chapter concludes with a description of multiplexing for dPCR using either fluorescently labelled probe-based detection techniques (as for qPCR) or DNA binding dyes. The fifth chapter investigates the effect of RNA quality control (in terms of purity and integrity) on PCR expression profiling. Following an introductory description of methods evaluate the RNA quality, the authors proceed with a presentation of UV/Vis spectrophotometry and 2100 Bio analyser components. Next, the RNA purity control using optical density measurement on nanodrop and total RNA and small RNA integrity control on 2100 Bio analyser are presented. The authors include details about an alternative test, the SPUD assay for qPCR inhibitors and the 18S/28S ratio agarose gel electrophoresis for measuring the total RNA integrity. The sixth chapter presents mediator probe PCR (MP PCR), a novel detection approach which uses label-free mediator probes and universal fluorogenic reporters (UR) oligonucleotides. It consists of a detailed introduction of this method followed by extensive details regarding the materials, the templates, the primers, enzymes and buffers. Next, the authors include a step by step description of the method, presenting in detail the choices and outcomes that can occur. The chapter concludes with a brief troubleshooting section that complements the extensive presentation from the previous sections. The seventh chapter presents the use of qPCR in the context of viral DNA quantification. It highlights the benefits of a calibrator technology to overcome the increase of the PCR artefacts and to control the efficiency of nucleic acid recovery during the DNA extraction phase. Following a brief description of the calibrated real-time PCR technique, the authors describe in detail the required materials. Next, each step of the methods is meticulously described and details on sample loading, RT-PCR run conditions and calculation of the recovery rate and DNA normalization are also included. The eighth chapter describes a multiplex RT-PCR platform developed for the rapid detection of oncogenic viral load. The required materials and primers for the detection of specific genotypes are included next to a detailed description of experimental steps. The chapter concludes with troubleshooting notes and a simple approach to normalize the HPV-type specific viral load. The ninth chapter describes a practical use for the detection of \textit{Mycoplasma pneumoniae} using Scorpion probe real-time PCR. As for the previous chapter, the required materials and experimental steps are described in detail. Chapter 10 shows yet another facet of qPCR used for the detection of impurities in cultured (human) cells. The authors present a multiplexed qPCR assay which allows the detection of quasi-species in a single-tube reaction using the intergenic spacers of 16S-23S rRNA and the Tuf and P1 cytoadhesin genes. Following the same detailed pattern of previous chapters, the authors describe an assay for monitoring BCR-ABL1 gene in chronic myeloid leukemia in Chapter 11 and for the identification of transplacental metastasis in Chapter 12. In Chapter 13 the authors focus on understanding and solving the issues around sample collection and extraction for the detection of circulating cell-free DNA (cfDNA) in cancer. A method for the accurate quantification of total cfDNA from plasma is presented in detail. Chapter 14 presents the use of qPCR on larger scale for gene expression analysis in clinical kidney transplantation. Chapter 15 extends the use of qPCR. The miRNA quantification approach is presented in detail and the differences from mRNA quantification are discussed. The chapter also includes a brief description of an integrative analysis of miRNA and mRNA expression data and its interpretation. Bottlenecks for the correct identification of miRNA targets in animals and approaches to circumvent these (such as enrichment GO term analysis) are also discussed. Chapter 16 presents the dPCR focusing on its clinical applications. The author discusses pathogen/viral detection and quantification, copy number variation analysis and rare mutation detection in detail. The materials and methods required for these tasks are meticulously described and the chapter concludes with an extensive series of notes aimed at helping the troubleshooting process. Another facet of the dPCR is presented in the last chapter where authors describe a non-invasive diagnosis method for single gene disorders. The book is a valuable collection of qPCR-related details that cover a wide range of PCR uses. Aimed mainly at researchers (wet lab biologists) with a solid PCR background, the book provides enough details for the novice too, who may find the numerous examples as well as the end notes in each chapter very useful.