rt-pcr protocols - joe o’connell

Methods in Molecular Biology TM HUMANA PRESS HUMANA PRESS Methods in Molecular Biology TM Edited by Joe O’Connell RT-PCR Protocols VOLUME 193 Edited by Joe O’Connell RT-PCR Protocols RT-PCR Protocols M E T H O D S I N M O L E C U L A R B I O L O G Y TM John M. Walker, S ERIES E DITOR 220. Cancer Cytogenetics: Methods and Protocols, edited by John Swansbury, 2003 219. Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications, edited by Joseph M. Metzger, 2003 218. Cancer Cell Signaling: Methods and Protocols, edited by David M. Terrian, 2003 217. Neurogenetics: Methods and Protocols, edited by Nicholas T. Potter, 2003 216. PCR Detection of Microbial Pathogens: Methods and Pro- tocols, edited by Konrad Sachse and Joachim Frey, 2003 215. Cytokines and Colony Stimulating Factors: Methods and Protocols, edited by Dieter Körholz and Wieland Kiess, 2003 214. Superantigen Protocols, edited by Teresa Krakauer, 2003 213. Capillary Electrophoresis of Carbohydrates, edited by Pierre Thibault and Susumu Honda, 2003 212. Single Nucleotide Polymorphisms: Methods and Protocols, edited by Piu-Yan Kwok, 2003 211. Protein Sequencing Protocols, 2nd ed., edited by Bryan John Smith, 2003 210. MHC Protocols, edited by Stephen H. Powis and Robert W. Vaughan, 2003 209. Transgenic Mouse Methods and Protocols, edited by Mar- ten Hofker and Jan van Deursen, 2002 208. Peptide Nucleic Acids: Methods and Protocols, edited by Peter E. Nielsen, 2002 207. Recombinant Antibodies for Cancer Therapy: Methods and Protocols. edited by Martin Welschof and Jürgen Krauss, 2002 206. Endothelin Protocols, edited by Janet J. Maguire and Anthony P. Davenport, 2002 205. E. coli Gene Expression Protocols, edited by Peter E. Vaillancourt, 2002 204. Molecular Cytogenetics: Protocols and Applications, edited by Yao-Shan Fan, 2002 203. In Situ Detection of DNA Damage: Methods and Protocols, edited by Vladimir V. Didenko, 2002 202. Thyroid Hormone Receptors: Methods and Protocols, edited by Aria Baniahmad, 2002 201. Combinatorial Library Methods and Protocols, edited by Lisa B. English, 2002 200. DNA Methylation Protocols, edited by Ken I. Mills and Bernie H, Ramsahoye, 2002 199. Liposome Methods and Protocols, edited by Subhash C. Basu and Manju Basu, 2002 198. Neural Stem Cells: Methods and Protocols, edited by Tanja Zigova, Juan R. Sanchez-Ramos, and Paul R. Sanberg, 2002 197. Mitochondrial DNA: Methods and Protocols, edited by Will- iam C. Copeland, 2002 196. Oxidants and Antioxidants: Ultrastructure and Molecular Biology Protocols, edited by Donald Armstrong, 2002 195. Quantitative Trait Loci: Methods and Protocols, edited by Nicola J. Camp and Angela Cox, 2002 194. Posttranslational Modifications of Proteins: Tools for Func- tional Proteomics, edited by Christoph Kannicht, 2002 193. RT-PCR Protocols, edited by Joe O’Connell, 2002 192. PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen and Harry W. Janes, 2002 191. Telomeres and Telomerase: Methods and Protocols, edited by John A. Double and Michael J. Thompson, 2002 190. High Throughput Screening: Methods and Protocols, edited by William P. Janzen, 2002 189. GTPase Protocols: The RAS Superfamily, edited by Edward J. Manser and Thomas Leung, 2002 188. Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 187. PCR Mutation Detection Protocols, edited by Bimal D. M. Theophilus and Ralph Rapley, 2002 186. Oxidative Stress Biomarkers and Antioxidant Protocols, edited by Donald Armstrong, 2002 185. Embryonic Stem Cells: Methods and Protocols, edited by Kursad Turksen, 2002 184. Biostatistical Methods, edited by Stephen W. Looney, 2002 183. Green Fluorescent Protein: Applications and Protocols, edited by Barry W. Hicks, 2002 182. In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Braman, 2002 181. Genomic Imprinting: Methods and Protocols, edited by Andrew Ward, 2002 180. Transgenesis Techniques, 2nd ed.: Principles and Proto- cols, edited by Alan R. Clarke, 2002 179. Gene Probes: Principles and Protocols, edited by Marilena Aquino de Muro and Ralph Rapley, 2002 178. Antibody Phage Display: Methods and Protocols, edited by Philippa M. O’Brien and Robert Aitken, 2001 177. Two-Hybrid Systems: Methods and Protocols, edited by Paul N. MacDonald, 2001 176. Steroid Receptor Methods: Protocols and Assays, edited by Benjamin A. Lieberman, 2001 175. Genomics Protocols, edited by Michael P. Starkey and Ramnath Elaswarapu, 2001 174. Epstein-Barr Virus Protocols, edited by Joanna B. Wilson and Gerhard H. W. May, 2001 173. Calcium-Binding Protein Protocols, Volume 2: Methods and Techniques, edited by Hans J. Vogel, 2001 172. Calcium-Binding Protein Protocols, Volume 1: Reviews and Case Histories, edited by Hans J. Vogel, 2001 171. Proteoglycan Protocols, edited by Renato V. Iozzo, 2001 170. DNA Arrays: Methods and Protocols, edited by Jang B. Rampal, 2001 169. Neurotrophin Protocols, edited by Robert A. Rush, 2001 168. Protein Structure, Stability, and Folding, edited by Ken- neth P. Murphy, 2001 167. DNA Sequencing Protocols, Second Edition, edited by Colin A. Graham and Alison J. M. Hill, 2001 166. Immunotoxin Methods and Protocols, edited by Walter A. Hall, 2001 165. SV40 Protocols, edited by Leda Raptis, 2001 164. Kinesin Protocols, edited by Isabelle Vernos, 2001 163. Capillary Electrophoresis of Nucleic Acids, Volume 2: Practical Applications of Capillary Electrophoresis, edited by Keith R. Mitchelson and Jing Cheng, 2001 162. Capillary Electrophoresis of Nucleic Acids, Volume 1: Introduction to the Capillary Electrophoresis of Nucleic Acids, edited by Keith R. Mitchelson and Jing Cheng, 2001 161. Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001 160. Nuclease Methods and Protocols, edited by Catherine H. Schein, 2001 Humana Press Totowa, New Jersey M E T H O D S I N M O L E C U L A R B I O L O G Y™ RT-PCR Protocols Edited by Joe O’Connell Department of Medicine, National University of Ireland, Cork, Ireland ©2002 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology ™ is a trademark of The Humana Press Inc. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Production Editor: Kim Hoather-Potter. Cover design by Patricia F. Cleary. Cover illustration: Background from Fig. 3A in Chapter 17 “RT-PCR-Based Approaches to Generate Probes for mRNA Detection by In Situ Hybridization” by Joe O’Connell; foreground from Fig. 2 in Chapter 18 “Amplified RNA for Gene Array Hybridizations” by Valentina I. Shustova and Stephen J. Meltzer. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: humana@humanapr.com or visit our Website: http://humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-875-0/02 $10.00 + $00.25]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data RT-PCR Protocols/edited by Joseph O’Connell p.cm (Methods in molecular biology) Includes bibliographical references and index. ISBN 0-89603-875-0 (alk. paper) 1. Polymerase chain reaction Laboratory manuals. I. O’Connell, Joseph. II. Methods in molecular biology (Clifton, N.J.); v. 193. QP606.D46 R8 2002 572'.43 dc21 2002190221 v Preface Until the mid 1980s, the detection and quantification of a specific mRNA was a difficult task, usually only undertaken by a skilled molecular biologist. With the advent of PCR, it became possible to amplify specific mRNA, after first converting the mRNA to cDNA via reverse transcriptase. The arrival of this technique—termed reverse transcription-PCR (RT-PCR)—meant that mRNA suddenly became amenable to rapid and sensitive analysis, without the need for advanced training in molecular biology. This new accessibility of mRNA, which has been facilitated by the rapid accumulation of sequence data for human mRNAs, means that every biomedical researcher can now include measurement of specific mRNA expression as a routine component of his/her research plans. In view of the ubiquity of the use of standard RT-PCR, the main objective of RT-PCR Protocols is essentially to provide novel, useful applications of RT-PCR. These include some useful adaptations and applications that could be relevant to the wider research community who are already familiar with the basic RT-PCR protocol. For example, a variety of different adaptations are described that have been employed to obtain quantitative data from RT-PCR. Quantitative RT-PCR provides the ability to accurately measure changes/imbal- ances in specific mRNA expression between normal and diseased tissues. Because of its remarkable sensitivity, RT-PCR enables the detection of low-abun- dance mRNAs even at the level of individual cells. RT-PCR has afforded many opportunities in diagnostics, allowing sensitive detection of RNA viruses such as HIV and HCV. RT-PCR facilitates many diverse techniques in research, includ- ing in situ localization of mRNA, antibody engineering, and cDNA cloning. In particular, the present work highlights how RT-PCR complements other tech- nological advances, such as laser-capture microdissection (LCM), real-time PCR, microarray technology, HPLC, and time-resolved fluorimetry. RT-PCR has become one of the most widely applied techniques in bio- medical research, and has been a major boon to the molecular investigation of disease pathogenesis. Determination of the pathogenesis of diseases at the molecular level is already beginning to inform the design of new therapeutic strategies. It is our hope that RT-PCR Protocols will stimulate the reader to explore diverse new ways in which this remarkable technique can facilitate the molecular aspects of their biomedical research. Joe O’Connell vii Contents Preface v Contributors xi PART I. INTRODUCTION 1 RT-PCR in Biomedicine: Opportunities Arising from the New Accessibility of mRNA Joe O’Connell 3 2 The Basics of RT-PCR: Some Practical Considerations Joe O’Connell 19 PART II. HIGHLY SENSITIVE DETECTION AND ANALYSIS OF MRNA 3 Using the Quantitative Competitive RT-PCR Technique to Analyze Minute Amounts of Different mRNAs in Small Tissue Samples Susanne Greber-Platzer, Brigitte Balcz, Christine Fleischmann, and Gert Lubec 29 4 Detection of mRNA Expression and Alternative Splicing in a Single Cell Tsutomu Kumazaki 59 5 Nested RT-PCR: Sensitivity Controls are Essential to Determine the Biological Significance of Detected mRNA Triona Goode, Wen-Zhe Ho, Terry O’Connor, Sandra Busteed, Steven D. Douglas, Fergus Shanahan, and Joe O’Connell 65 PART III. QUANTITATIVE RT-PCR 6 Quantitative RT-PCR: A Review of Current Methodologies Caroline Joyce 83 7 Rapid Development of a Quantitative-Competitive (qc) RT-PCR Assay Using a Composite Primer Approach Joe O’Connell, Aileen Houston, Raymond Kelly, Darren O’Brien, Aideen Ryan, Michael W. Bennett, and Kenneth Nally 93 8 Quantitation of Gene Expression by RT-PCR and HPLC Analysis of PCR Products Franz Bachmair, Christian G. Huber, and Guenter Daxenbichler 103 viii Contents 9 Time-Resolved Fluorometric Detection of Cytokine mRNAs Amplified by RT-PCR Kaisa Nieminen, Markus Halminen, Matti Waris, Mika Mäkelä, Johannes Savolainen, Minna Sjöroos, and Jorma Ilonen 117 10 Mimic-Based RT-PCR Quantitation of Substance P mRNA in Human Mononuclear Phagocytes and Lymphocytes Jian-Ping Lai, Steven D. Douglas, and Wen-Zhe Ho 129 PART IV. DETECTION AND ANALYSIS OF RNA VIRUSES 11 Detection and Quantification of the Hepatitis C Viral Genome Liam J. Fanning 151 12 Semi-Quantitative Detection of Hepatitis C Virus RNA by "Real-Time" RT-PCR Joerg F. Schlaak 161 13 RT-PCR for the Assessment of Genetically Heterogenous Populations of the Hepatitis C Virus Brian Mullan, Liam J. Fanning, Fergus Shanahan, and Daniel G. Sullivan 171 PART V. I N S ITU LOCALIZATION OF MRNA EXPRESSION 14 In Situ Immuno-PCR: A Newly Developed Method for Highly Sensitive Antigen Detection In Situ Yi Cao 191 15 RT-PCR from Laser-Capture Microdissected Samples Tatjana Crnogorac-Jurcevic, Torsten O. Nielsen, and Nick R. Lemoine 197 16 Mycobacterium paratuberculosis Detected by Nested PCR in Intestinal Granulomas Isolated by LCM in Cases of Crohn’s Disease Paul Ryan, Simon Aarons, Michael W. Bennett, Gary Lee, Gerald C. O’Sullivan, Joe O’Connell, and Fergus Shanahan 205 17 RT-PCR-Based Approaches to Generate Probes for mRNA Detection by In Situ Hybridization Joe O’Connell 213 PART VI. DIFFERENTIAL MRNA EXPRESSION 18 Amplified RNA for Gene Array Hybridizations Valentina I. Shustova and Stephen J. Meltzer 227 19 Semi-Quantitative Determination of Differential Gene Expression in Primary Tumors and Matched Metastases by RT-PCR: Comparison with Other Methods Benno Mann and Christoph Hanski 237 PART VII. GENETIC ANALYSIS 20 Detection of Single Nucleotide Polymorphisms Using a Non-Isotopic RNase Cleavage Assay Frank Waldron-Lynch, Claire Adams, Michael G. Molloy, and Fergal O’Gara 253 PART VIII. RT-PCR IN IMMUNOLOGY 21 Detection of Clonally Expanded T-Cells by RT-PCR-SSCP and Nucleotide Sequencing of T-Cell Receptor β-CDR3 Regions Manae Suzuki Kurokawa, Kusuki Nishioka, and Tomohiro Kato 267 22 Generation of scFv from a Phage Display Mini-Library Derived from Tumor-Infiltrating B-Cells Nadège Gruel, Beatrix Kotlan, Marie Beuzard, and Jean-Luc Teillaud 281 23 Generation of Murine scFv Intrabodies from B-Cell Hybridomas Chang Hoon Nam, Sandrine Moutel, and Jean-Luc Teillaud 301 24 Quantitation of mRNA Levels by RT-PCR in Cells Purified by FACS: Application to Peripheral Cannabinoid Receptors in Leukocyte Subsets Jean Marchand and Pierre Carayon 329 PART IX. RT-PCR IN ANTI-SENSE TECHNOLOGY 25 Detection of Anti-Sense RNA Transcripts by Anti-Sense RT-PCR Michael C. Yeung and Allan S. Lau 341 PART X. RT-PCR IN CDNA CLONING 26 RT-PCR in cDNA Library Construction Vincent Healy 349 27 An RT-PCR-Based Protocol for the Rapid Generation of Large, Representative cDNA Libraries for Expression Screening Joe O’Connell 363 Index 375 Contents ix [...]... for the protocols described, so that even a newcomer to RT-PCR should be able to perform the techniques In particular, this volume demonstrates how RT-PCR complements other technologies, such as laser-capture microdissection (LCM), real-time PCR, microarray analysis, high-pressure liquid chromatography (HPLC) and time-resolved fluorometry From: Methods in Molecular Biology, vol 193: RT-PCR Protocols. .. The Basics of RT-PCR 19 2 The Basics of RT-PCR Some Practical Considerations Joe O’Connell 1 Introduction The basic reverse-transcriptase-polymerase chain reaction (RT-PCR) technique is used routinely and widely in most biomedical research laboratories The fundamental considerations for such basics as primer design, good laboratory set up and practice, and techniques for performing RT-PCR have been... FRANK WALDRON-LYNCH • University of Cambridge Clinical School, Addenbrooke’s Hospital, Cambridge, UK MATTI WARIS • Laboratory of Biophysics, University of Turku, Finland MICHAEL C YEUNG • Snyder Research Foundation, Winfield, KS RT-PCR in Biomedicine I INTRODUCTION 1 2 O'Connell RT-PCR in Biomedicine 3 1 RT-PCR in Biomedicine Opportunities Arising from the New Accessibility of mRNA Joe O’Connell 1 Introduction... significantly from RT-PCR; the expression of low-abundance mRNAs can now be measured in small tissue samples from specific areas of the brain (3,4) In Chapter 3, for example, RT-PCR is applied to detect and quantify specific mRNA at the femto/attogram level in minute amounts of brain tissue Although the extraordinary sensitivity of nested RT-PCR is a huge advantage for the detection of low-abundance mRNA,... pertaining to the use and interpretation of data from nested RT-PCR; unless a quantitative approach is employed, sensitivity controls should be adopted to estimate the level of mRNA detected by the nested RT-PCR assay Otherwise, the amount of detected mRNA can be overestimated (5) 3 Quantitative RT-PCR: Approaches and Applications The sensitivity of RT-PCR makes it particularly useful for detecting lowabundance... application of RT-PCR; in addition to providing a highly sensitive diagnostic test, quantitative RT-PCR tests enable the viremia level to be monitored in response to therapy RT-PCR also provides material for genotype analysis of the virus present, and allows the presence and sequence diversity of variant viral “quasispecies” to be analyzed (11) (see Chapter 13) Also useful in virology research, RT-PCR can... process, RT-PCR has many other applications in cancer research and diagnosis RT-PCR for mRNAs encoding various tumor markers, including carcinoembryonic antigen (CEA), has frequently been used to detect the presence of tumor micrometastases in patient bone marrow and in the circulation (18) RT-PCR also facilitates the detection of mutations in oncogenes or tumor-suppressor genes, such as APC and BRCA-1,... denaturant—such as dimethyl sulfoxide RT-PCR in Biomedicine 15 (DMSO) or formamide—in the PCR appears to minimize such bias, presumably by “smoothing out” secondary structures and denaturing (GC)-rich regions that give rise to poor amplification efficiency in some cDNAs RT-PCR is particularly useful in the construction of cDNA libraries when the source of cell or tissue is limited; RT-PCR- based cDNA libraries have... greatest advantage of RT-PCR in the analysis of mRNA is its extraordinary sensitivity Using nested RT-PCR, mRNA can essentially be detected at the level of single copies Many of the chapters in this volume demonstrate the highly sensitive detection of mRNA In Chapter 4, nested RT-PCR is used to analyze mRNA expression in a single cell; a single cell is lysed and placed directly in the RT-PCR reaction Using... Biology, vol 193: RT-PCR Protocols Edited by:J O'Connell © Humana Press Inc., Totowa, NJ 19 20 O'Connell out DNA contamination is probably by ultracentrifugation of RNA lysates through cesium chloride density gradients (4), a technique that is obviously not suited to routine requirements for RT-PCR The simplest solution to this problem is the use of intron-spanning primers for the RT-PCR, so that PCR . PRESS HUMANA PRESS Methods in Molecular Biology TM Edited by Joe O’Connell RT-PCR Protocols VOLUME 193 Edited by Joe O’Connell RT-PCR Protocols RT-PCR Protocols M E T H O D S I N M O L E C U L A R B I O. 329 PART IX. RT-PCR IN ANTI-SENSE TECHNOLOGY 25 Detection of Anti-Sense RNA Transcripts by Anti-Sense RT-PCR Michael C. Yeung and Allan S. Lau 341 PART X. RT-PCR IN CDNA CLONING 26 RT-PCR in cDNA. Shanahan, and Joe O’Connell 65 PART III. QUANTITATIVE RT-PCR 6 Quantitative RT-PCR: A Review of Current Methodologies Caroline Joyce 83 7 Rapid Development of a Quantitative-Competitive (qc) RT-PCR Assay