Donnerstag, 1. Juli 2010

Real-Time PCR: Reference Genes & Data Analysis

Real-Time PCR: Current Technology and Applications
Publisher: Caister Academic PressEditor: Julie Logan, Kirstin Edwards and Nick Saunders Applied and Functional Genomics, Health Protection Agency, London (2009) ISBN: 978-1-904455-39-4

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