1st Edition

PCR/RT- PCR in situ Light and Electron Microscopy

By Gerard Morel, Mireille Raccurt Copyright 2002
    444 Pages 21 Color & 237 B/W Illustrations
    by CRC Press

    Although the polymerase chain reaction has revolutionized genetic analysis by amplifying rare nucleic acid sequences, the in situ application is the only method that allows the localization of amplified signal within tissue structure. The applications of in situ polymerase chain reaction have greatly enhanced the field of investigation in many disciplines, including viral infections, gene modification, tumor diagnosis, gene therapy, and cellular distribution of rare mRNA copies.

    PCR/RT-PCR in situ: Light and Electron Microscopy covers methods of in situ polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), two new approaches in visualizing very low amounts of DNA and RNA in tissues and cell cultures at the light and electron microscopy levels. Written by experts in this field, the book provides theoretical consideration, as well as practical approaches to in situ PCR. The authors provide detailed protocols for each step, including the preparation of tissue samples, the rationale for the design of primers and revelation. They also emphasize the need for appropriate controls to meet the requirements of in situ PCR and RT-PCR specificity. Organized in a user-friendly two-column format, this book will provide you with tools necessary to perform and optimize these sensitive and powerful techniques in your research protocols.

    GENERAL PRINCIPLES
    PREPARATION OF SAMPLES
    PRETREATMENTS
    REVERSE TRANSCRIPTION (RT)
    POLYMERASE CHAIN REACTION (PCR)
    HYBRIDIZATION
    REVELATION
    ELECTRON MICROSCOPY
    CONTROLS AND PROBLEMS
    TYPICAL PROTOCOLS
    EXAMPLES OF OBSERVATIONS
    APPENDICES
    A-Equipment
    B-Reagents
    GLOSSARY
    INDEX

    Short TOC
    General Principles. Preparation of Samples. Pretreatments. Reverse Transcription (RT). Polymerase Chain Reaction (PCR). Hybridization. Revelation. Electron Microscopy. Controls and Problems. Typical Protocols. Examples of Observations. Appendices. Glossary. Index

    Biography

    GĂ©rard Morel, Ph.D., D.Sc., is a research director at the National Center of Scientific Research (CNRS), at University Claude Bernard-Lyon 1, Villeurbanne, France. Mireille Raccurt, Ph.D., is an engineer in biology in a laboratory of the CNRS (Molecular Physiology), at Claude Bernard-Lyon 1 University, Villeurbanne, France.