Quantitative PCR Protocols - Bernd Kochanowski

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Résumé :
Since the polymerase chain reaction (PCR) was first developed in 1985, an enormous number of research reports have documented the versatility of this brilliant technique for in vitro amplification of nucleic acids. Although PCR has had a profound impact in many areas of research, contrary to exp- tation its routine application to the quantitation of nucleic acids has proven problematic in several aspects. The shortcomings are principally caused by the exponential nature of PCR, whereby small variations in amplification ef- ciency may dramatically affect the yield of amplification product. Even mi- mal temperature deviations that occur between adjacent wells of a thermocycler or day-to-day variations in the efficiency of nucleic acid preparation can lead to significant differences in the extent of amplification between otherwise id- tical samples. However, knowing more about the intrinsic limitations of PCR is the first step towards surmounting the shortcomings associated with this prom- ing methodology. With the introduction of appropriate standards of known amount, which are co-amplified with the sample using the same primers, it is increasingly feasible to address biological or diagnostic questions that are d- ficult or impossible to answer using any other experimental approach.

Sommaire:
Part I. Reviews Quantitative PCR: A Survey of the Present Technology Udo Reischl and Berndt Kochanowski General Principles of Quantitative PCR Luc Raeymaekers Effects of Collection, Processing, and Storage on RNA Detection and Quantification Mark Holodniy Quantitative RT-PCR Paul D. Siebert Kinetic Quantitative PCR vs End-Point Quantitative PCR with Internal Standard Olivier Lantz, Elizabeth Bonney, Franck Griscelli, and Yassine Taoufik Comparison of Competitive PCR and Positive Control-Based PCR Fran?ois Mallet Part II. Protocols End-Point Titration-PCR for Quantitation of Cytomegalovirus DNA Jerzy K. Kulski Analysis of Amplified DNA Molecules by Capillary Electrophoresis and Laser Induced Fluorescence Michael J. Fasco Competitor Calibration and Analysis of Competitive Amplified PCR Products by High-Performance Liquid Chromatography (HPLC) Thomas KÖhler Quantifying Amplicons with ELISA Olivier Lantz, Elizabeth Bonney, and Yassine Taoufik Competitive PCR Quantitation Utilizing a Microtiter Plate Based Format for the Detection of PCR Products Bernd Kochanowski and Wolfgang Jilg Competitive and Differential RT-PCR (CD-RT-PCR) for Measurement of Normalized Gene Expression Using Antisense Competitors Eric de Kant Amplified Assay for Specific Dual-Labeled DNA Using the Coagulation Cascade (EDNA-ELCA) George J. Doellgast, G. Alan Beard, Margaret Sheehan, Nathan Iyer, Louis S. Kucera, and Stephen H. Richardson Quantitative PCR with Internal Standardization and OLA-ELISA Product Analysis for the p53 Tumor Suppressor Gene Meinhard Hahn and Alfred Pingoud Quantitative Analysis of Human DNA Sequences by PCR and Solid-Phase Minisequencing Anu Suomalainen and Ann-Christine SyvÄnen High Resolution PCR Quantitation byAmpliSensor Assay Chang-Ning Wang Construction of Polycompetitors for Competitive PCR David B. Corry and Richard M. Locksley Tailed RT-PCR for the Quantitation of Chloramphenicol Acetyl Transferase (CAT)mRNA Marlyse C. Knuchel and Aftab A. Ansari A Stochastic PCR Approach for RNA Quantification in Multiple Samples Adrian Puntschart and Michael Vogt Quantitation of mRNA Species by RT-PCR on Total mRNA Population with Nonradioactive Probes Sabine Herblot, Beno?t Rousseau, and Jacques Bonnet Index