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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetic Improvement of Wheat and Barley for Resistance to Biotic and Abiotic Stresses

Location: Wheat Genetics, Quality Physiology and Disease Research

Title: Real-time PCR: Advanced technologies and applications

Author
item Skinner, Daniel

Submitted to: Crop Science
Publication Type: Review Article
Publication Acceptance Date: October 3, 2013
Publication Date: December 13, 2014
Repository URL: http://doi:10.2135/cropsci2013.12.0001br
Citation: Skinner, D.Z. 2014. Real-time PCR: Advanced technologies and applications. Crop Science. 24:1:455-456.

Interpretive Summary: This review of the book “Real-Time PCR: Advanced Technologies and Applications” provides a summary of the information presented in the book, with interpretation of how it may be useful to researchers during the planning and design of investigations that may benefit from real-time PCR. This review will enable readers to move immediately to the specific chapters of the book that have the information needed to assist in each phase of an investigation.

Technical Abstract: This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide the novice and the experienced user with an invaluable point of reference for this technology. They have succeeded in reaching this goal and in so doing, the book has, perhaps inadvertently, provided a powerful guide to use in designing qPCR experiments. The first chapter begins the book with a thorough discussion of fluorescent chemistries for amplicon detection and their advantages and disadvantages. Beginning with simple DNA-binding dyes and progressing through nuclease activity-based probes, to sequence-specific probes, to self-probing amplicons, the authors provide invaluable details of how these options function and what results may be expected. In addition, this chapter discussed specific machines available and their capabilities, and dedicates a section to intellectual property concerns and how that may need to be considered as one prepares to conduct qPCR experiments. As the design of an experiment continues, Chapters 2, 7, 8, and 11 can be consulted for valuable information on methods and options. Chapter 2 provides a thorough discussion of internal and external controls. Creative design and use of “molecular mimic” internal controls is described with easily understood diagrams and examples of results. Chapter 7 provides a succinct but thorough review of the methods that have been used to isolate nucleic acids and prepare them for PCR, and Chapter 8 discusses a number of ways to “fine-tune” the specificity of primers using chemical modifications. Chapter 11 provides a discussion of the “minimum standard for the provision of information for qPCR experiments (MIQE).” Chapter 11 is only nine pages long but provides a wealth of guidance on what steps to take to ensure that qPCR results are sound, reproducible, and obtained in such a way that the underlying hypotheses can be tested reliably. Chapter 3 takes the reader through the “nuts and bolts” of performing a qPCR experiment, including sample preparation and quality assessment, PCR setup, data generation, and data analysis. Potential pitfalls and ways to avoid them are also discussed, and several pages and examples are dedicated to optimization of the PCR. Chapters 9 and 10 are dedicated to qPCR arrays and their capabilities, constraints, and validation. The background information provided here is sure to be of interest to anyone who is contemplating assaying hundreds of genes, and the suggestions presented in Chapter 10 for validating the results should be useful to anyone hoping to use PCR arrays to relate phenotypes to qPCR results. Chapters 4-6 provide “real world” examples of using qPCR to detect and quantify microorganisms in human and veterinary medicine, as well as a detection tool in possible biowarfare applications. These chapters provide food for thought for possible applications in crop agriculture. The book concludes with a chapter entitled “Management aspects of real-time PCR-based assay development, validation, verification and implementation.” The guidance provided in this chapter, as the title implies, is extensive. While its recommendations in their entirety are daunting, and are geared toward qPCR applications to clinical diagnosis and research, one would be well-advised to become familiar with the tenets of this chapter. Following these recommendations would lead to well-structured, documented, and verifiable data from any qPCR experiment. Anyone considering extensive use of qPCR should read this book to become familiar with the multitude of approaches and materials available. Applying the information provided at the outset of an experiment will greatly improve the chances of reproducibility and success.

Last Modified: 10/24/2014
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