RNA Isolation and

Characterization 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 "

John M. Walker, SERIES EDlrOR

102. Biolumineseenee Methods and Protocols, edited by Robert A. LaRossa, 1998

101. Myobacteria Protocols, edited by Tanya Parish and Neil G. Stoker, 1998

100. Nitric Oxide Protocols, edited by ~ A. Titheradge, 1997 99. Human Cytokines and Cytokine Receptors, edited by Reno

Debets, 1998 98. DNA Profiling Protocols, edited by James M. Thomson, 1998 97. Molecular Embryology: Methods and Protocols, edited by

Paul T. Sharpe, 1998 96. Adhesion Proteins Protocols, edited by Elisabetta Dejana,

1998 95. DNA Topology and DNA Topoisomerases: II. Enzymology

and Topoisomerase Targetted Drugs, edited by Many-Ann Bjornsti, 1998

94, DNA Topology and DNA Topoisomerases: L ONA Topology and Enzyme Purification, edited by Mary-Ann Bjornsti, 1998

93. Protein Phospbatase Protocols, edited by John W. Ludlow, 1998 92. PCR in Bioanalysis, edited by Stephen Meltzer, 1997 91. Flow Cytometry Protocols, edited by MarkJ. Jaroszeski, 1998 90. Drug-DNA Interactions: Methods, Case Studies, and Proto-

cols, edited by Keith R. Fox, I997 89. Retinoid Protocols, edited by Christopher Redfern, 1997 88. Protein Targeting Protocols, edited by Roger A. Clegg, 1997 87. Combinatorial Peptide Library Protocols, edited by Shmuel

Cabilly, 1997 86. RNA Isolation and Characterization Protocols, edited by

Ralph Rapley and David L. Manning, 1998 85. Differential Display Methods and Protocols, edited by Peng

Liang and Arthur B. Pardee, 1997 84. Transmembrane Signaling Protocols, edited by Dafna Bar-

Sagi, 1997 83. Receptor Signal Transduetion Protocols, edited by R. A. J.

Challiss, 1997 82. Arabidopsis Protocols, edited by Jos~ M Martinez-Zapater and

dulio Salinas, I998 81. Plant Virology Protocols, edited by Gary D. Foster, 1998 80. Immuuoehemieal Protocols, secouD EDITION, edited by John

Pound, 1998 79. Polyamine Protocols, edited by David M. L. Morgan, 1998 78. Antibacterial Peptide Protocols, edited by William M. Shafer,

1997 77. Protein Synthesis: Methods and Protocols, edited by Robin

Martin, 1998 76. Glyeoaualysis Protocols, edited by Elizabeth F. Hounsell, 1998 75. Basic Cell Culture Protocols, edited by Jeffrey W. Pollard

and John M. Walker, 1997 74. Ribozyme Protocols, edited by Philip C. Turner, 1997 73. Neuropeptide Protoenls, edited by G. Brent 1trine and Carvell

Williams, 1997 72. Neurotransmitter Methods, edited by Richard C. Rayne, 1997 71. PR1NS and In Situ PCR Protocols, edited by John R. Gosden,

1997

70. Sequence Data Analysis Guidebook, edited by Simon R. Swindell, 1997

69. eDNA Library Protocols, edited by lan G. Cowell and Caroline A. Austin, 1997

68. Gene Isolation and Mapping Protocols, edited by Jacqueline Boultwood, 1997

67. PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, edited by Bruce A. White, 1996

66. Epitope Mapping Protocols, edited by Glenn E. Morris, 1996 65. PCR Sequencing Protocols, edited by Ralph Rapley, 1996 64. Protein Sequencing Protocols, edited by Bryan ,~ Smith, 1996 63. Recombinant Proteins: Detection and Isolation Protocols, ed-

ited by Rocky S. Tuan, 1996 62. Recombinant Gene Expression Protocols, edited by Rocky

S. Tuan, 1996 61. Prutein and Peptide Analysis by Mass Spectrometry, ed-

ited by John R. Chapman, 1996 60. Protein NMR Protocols, edited by David G. Reid, 1996 59. Protein Purification Protocols, edited by Shawn Doonan

1996 58. Basic DNA and RNA Protocols, edited byAdriand. Harwooa

I996 57. In Vitro Mutagenesis Protocols, edited by MichaelK. Trower

1996 56. Crystallographic Methods and Protocols, edited by Chris-

topher Jones, Barbara Mulloy, and Mark Sanderson, 1996 55. Plant Cell Electroporation and Eleetrofnsion Protocols, ed-

itedby Jac A. Nickoloff •995 54. YAC Protocols, edited by David Markie, I995 53. Yeast Protocols: Methods in Cell and Molecular Biology,

edited by lvor t7. Evans, 1996 52. Capillary Eleetrophnresis: Principles, Instrumentation, and

Applications, edited by Kevin D. Altria, 1996 51. Antibody Engineering Protocols, edited by Sudhir Paul, 1995 50. Species Diagnostics Protocols: PCR and Other Nucleic Acid

Methods, edited by Justin P. Clapp, t996 49. Plant Geue Transfer and Expression Protocols, edited by

Heddwyn Jones, 1995 48. Animal Cell Electroporation and Eleetrofusion Protocols,

edited by Jac A. Nickoloff 1995 47. Eleetroporation Protocols for Microorganisms, edited by Jac

A. Nickoloff 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny Howard

and David M. Whitcombe, 1995 45. Monoelonal Antibody Protocols, edited by William C. Davis,

1995 44. Agrobaeterium Protocols, edited by Kevan M. A. Gartland and

Michael R. Davey, 1995 43. In Vitro Toxicity Testing Protocols, edited by Sheila O'Hare

and Chris K. Atterwill, 1995 42. ELISA: Theory and Practice, by John R. Crowther, 1995 41. Signal Transduetion Protocols, edited by David A. Kendall

and Stephen J. Hill 1995

RNA Isolation and

C h a racte rizat i o n Protocols

Edited by

Ralph Rapley University of Hertfordshire, Hatfield, UK

and

David L. Manning Tenovus Institute for Cancer Research, Cardiff UK

Humana Press ~ Totowa, New Jersey

© 1998 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

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Library of Congress Cataloging in Publication Data

RNA isolation and characterization protocols/edited by Ralph Rapley, David L. Manning. p. cm. - (Methods in molecular biologyT~; 86)

Includes index. ISBN 0-89603-494-l (hard: alk. paper).- ISBN 0-89603-393-7 (comb: alk. paper) 1. RNA-Purifieation-Laboratory manuals. 2. RNA-Analysis-Laboratory manuals.

I. Rapley, Ralph. II. Manning, David L. IlL Series: Methods in molecular biology (Clifton, NJ); 86.

QP623.R575 1998 572.8'8--dc21

Preface

Ribonucleic acids are central to cellular and molecular processes and perform vital functions in both structural and functional roles. RNA molecules form the bridge between the stable genetic information contained within DNA and enzymes and proteins that carry out much of the metabolism within the cell. Many of the sites of protein synthesis, the ribosomes within the cell, are composed of these ribonucleic acids as are the tRNA molecules that deliver the amino acid building blocks to the ribosomes. Of all the RNA species, the nucleic acid intermediate, messenger RNA, is a desirable source of material to biologists, since this reflects much of, what ultimately, is translated into enzymes and proteins. In order to determine the qualitative and quantitative changes in mRNA expression, a vast number of molecular biological techniques have been developed.

Key molecular methods that provide the means to initially isolate and analyze RNA molecules are the focus of this volume. In putting together this collection of protocols, we have tried to provide techniques that are most applicable and widely used. In particular, there are a number of isola- tion techniques included that have been developed, modified, or adapted to enable extraction from a variety of cell types, organisms, or subcellular organelles. Successful isolation of intact RNA is an essential starting point for any subse- quent analysis. This is why we have aimed to make this section comprehensive.

The analysis of RNA is the focus of the following chapters. It includes traditional methods of blotting and hybridization, through to those techniques such as differential display, which can measure changes in the expression of specific genes. Readers of this volume will see that many of the methods have been developed through the application of the polymerase chain reaction, which continues to be a profoundly influential technique used today. These and later chapters deal with transcription and translation in vitro and in situ visualiza- tion of RNA molecules.

All of the chapters are presented in the familiar style of the Methods in Molecular Biology T M series, with a short description of the basic theory of the technique and an outline of the procedure, followed by a Materials section listing all the reagents necessary for the protocol. The Methods section pro-

vi Preface

vides a full and comprehensive account of the protocol in a step-by-step series of actions. In addition, references to the notes provide valuable and useful pieces of information not found in traditional scientific papers, but which in many cases may mean the difference between success and failure of a particu- lar method. All contributors carry out their own research or lead groups that apply the techniques as a matter o f routine and therefore are best suited to providing such methods. In putting together RNA Isolation and Characteriza- tion Protocols, we would like to thank all the present authors that have taken the trouble and valuable time to prepare the individual chapters, Professor John Walker, the series editor for his helpful advice and guidance, and the staff at The Humana Press.

Ralph Rapley David L. Manning

Contents

Preface ................................................................................................. v Contributors ......................................................................................... ix

1 Introduction to Isolating RNA, Donald E. Macfarlane and Christopher E. Dahle ..................... 1

2 Large and Small Scale RNA Preparations from Eukaryotic Cells, Woifgang Uckert, Wolfgang Walther, and Ulrike Stein .......... 7

3 An Improved Rapid Method of Isolating RNA from Cultured Cells, David B. Batt, Gordon G. Carmichael, and Zhong Liu ......... 15

4 Isolating RNA with the Cationic Surfactant, Catrimox-14, Christopher E. Dahle and Donald E. Macfarlane ................... 19

5 RNA Extraction from Formalin-Fixed and Paraffin-Embedded Tissues,

Giorg i Stanta, Serena Bonin, and Rosella Perin ................... 23 6 Extraction and Purification of RNA from Plant Tissues Enriched

in Polysaccharides, Shu-Hua Cheng and Jeffrey R. Seemann ............................... 27

7 Isolation of Plant Mitochondriat RNA from Green Leaves, Fei Ye, Wol fgang O. Abel, and Ral f Resk i .............................. 33

8 Extraction of RNA from Fresh and Frozen Blood, Bima l D. M. Theoph i lus ............................................................. 39

9 Isolation of Total RNA from Bacteria, John Heptinstall ......................................................................... 47

10 Isolation of Total RNA from Tissues or Cell Lines: Visualization in Gel,

Tapas Mukhopadhyay and Jack A. Roth ................................ 55 11 Isolation of Messenger RNA,

Sian Bryant and David L. Manning .......................................... 61 12 UV Spectrometric Analysis of Ribonucleic Acids

Ralph Rapley and John Heptinstall ......................................... 65

viii

13

Contents

Formaldehyde Gel Electrophoresis of Total RNA, Sian Bryant and David L. Manning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

14 Preparation of RNA Dot-Blots, Rachel l-lodge ............................................................................. 73

15 Nonradioactive Northern Blotting, Rainer Low ................................................................................. 77

16 The Use of RNA Probes for the Analysis of Gone Expression: Northern Blot Hybridization and Ribonuclease Protection Assay,

Dominique Belin ........................................................................ 87 17 Analysis of RNA by Northern Blotting Using Riboprobes,

Rai Ajit K. Srivastava .............................................................. 103 18 RNA Quantitative Analysis from Fixed and Paraffin-Embedded

Tissues, Giorgi Stanta, Serena Bonin, and Rene Utrera .................... 113

19 Quantitative Analysis of RNA Species by PCR and Solid-Phase Minisequencing,

Anu Suomalainen and Ann-Christine Syvanen ................... 121 20 Preparation of Tissue Sections and Slides for mRNA

Hybridization, Giorgi Terenghi ........................................................................ 133

21 Detecting mRNA in Tissue Sections with Digoxigenin-Labeled Probes,

Giorgi Terenghi ........................................................................ 137 22 One-Tube RT-PCR with Sequence Specific Primes,

Ulr ich Proffer ............................................................................ 143 23 Identification of Differentially Exposed Genes by Nonradioactive

Differential Display Messenger RNA, Thomas C. G. Bosch and Jan U. Lohmann .......................... 153

24 Characterization of RNA Using Continuous RT-PCR Coupled with ELOSA,

Francois Mallet, Guy Oriol, and Bernard Mandrand ........... 161 25 Gone Expression Analysis by CD-RT-PCR,

Eric de Kant .............................................................................. 173 26 Primer Extension Analysis of mRNA,

Maggie Walmsley, Mark Leonard, and Roger Patient ......... 187

Contents ix

27 $1 Mapping Using Single-Stranded DNA Probes, Stephane Viville and Roberto Mantovani . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

28 Measurements of Rate of Transcription in Isolated Nuclie by Nuclear "Run-Off" Assay,

Rai Ajit K. Srivastava and Gustov Schonfeld ...................... 201 29 Transcription In Vitro Using Bacteriophage RNA Polymerases,

Elaine T. Schenhborn .............................................................. 209 30 In Vitro Translation of Messenger RNA in a Rabbit Reticulocyte

Lysate Cell-Free System, Louise O/liver and Charles D. B o y d ...................................... 221

31 In Vitro Translation of Messenger RNA in a Wheat Germ Extract Cell-Free System,

Louise O/liver, Anne Grobler-Rabie, Charles D. Boyd ....... 229 The Xenopus Egg Extract Translation System, Glenn M. Matthews and Alan Co/man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Purification and Characterization of Viral dsRNA Genome

Profiles by Crosshybridization, Lesley-Ann Martin and Peter P. C. Mer tens ......................... 249

Index ................................................................................................. 261

32

33

Contributors

DAVID B. BATT * Department of Microbiology, University of Connecticut Health Center, Farmington, CT

DOMINIQUE BELIN * Department of Pathology, University of Geneva Medical School, Geneva, Switzerland

SERENA BONIN * International Centre of Genetic Engineering and Biotechnology-Padriciano (Trieste), Department of Pathology, University of Trieste

THOMAS C. G. BOSCH • Institut der Universitat, Munchen, Germany CHARLES D. BOYD • University of Medicine and Dentistry of New Brunswick,

New Brunswick, NJ SIAN BRYANT • Tenovus CancGT Research Centre, University of Wales College

of Medicine, Heath Park, UK GORDON G. CARMICHAEL • Department of Microbiology, University

of Connecticut Health Center, Farmington, CT SHU-HUA CHENG • Department of Biochemistry, University of Nevada, Reno, NV ALAN COLMAN • School of Biochemistry, Birmingham University, Birmingham, UK CHRISTOPHER E. DAHLE * Department of Medicine, University of Iowa, Iowa

City, IA ERIC DE KANT • Academeic Hospital Utrecht, Department of Internal

Medicine, Ultrecht, The Netherlands ANNE GROBLER-RABIE • University of Medicine and Dentistry of New Brunswick,

New Brunswick, NJ JOHN HEPTINSTALL • Biosciences Group, School of Natural and Environmental

Sciences, Coventry University, Coventry, UK RACHEL HODGE • Department of Botany, University of Leicester, Leicester, UK MARK LEONARD * Developmental Biology Research Centre, Kings College,

University of London, UK ZHONG LIU • Department of Microbiology, University of Connecticut Health

Center, Farmington, CT J A N U. LOHMANN * Institut der Universitat, Munchen, Germany RAINER L o w • Botanisches Institute, Ruprecht-Karls-Universitat, Heidelberg,

Germany xi

xii Contributors DONALD E. MACFARLANE • Department of Internal Medicine, University

of Iowa, Iowa City, I0 FRANCOIS MALLET • Ecole Normale Superieure de Lyon, Lyon, France BERNARD MANDRAND • Ecole Normale Superieure de Lyon, Lyon, France DAVID L. MANNING • Tenovus Cancer Research Centre, University

of Wales College of Medicine, Heath Park, UK ROBEWrO MANTOVAM • DOI/LGME Faculte de Medicine, Strasbourg, Cedex,

France LESLEY A N y M_~TIN • ICRF Oncology, Royal Postgraduate Medical School,

Surrey, UK GLEN MATa'H~WS • Department of Surgery, Queen Elizabeth Hospital,

Birmingham, UK PETER MERa'ENS • Institute for Animal Health, Pirbright,

Surrey, UK TAPAS MUKHOPADHYAY • Department of Thoracic and Cardiovascular Surgery,

The University of Texas M. D. Anderson Cancer Center, Houston, TX

L o u i s e OLLWER • University of Medicine and Dentistry of New Brunswick, New Brunswick, NJ

G u y OmOL • Ecole Normale Superieure de Lyon, Lyon, France ROG£R PATIENT • Developmental Biology Research Centre, Kings College,

University of London, UK ROSELLA PERIN • International Centre of Genetic Engineering

and Biotechnology-Padriciano (Trieste), Department of Pathology, University of Trieste

ULRICH PFEFFER • Laboratory of Molecular Biology, National Institute of Genoa, Genoa, Italy

RALPh RAPLEY • University of Hertfordshire, Hatfield, UK RALF REsr~ • Institute for General Botany, University of Hamburg, Hamburg,

Germany JACK A. Roa~-[ • Department of Thoracic and Cardiovascular Surgery, The University

of Texas M. D. Anderson Cancer Center, Houston, TX ELAINE T. SCHENBORN • Promega Corporation, Madison, WI G u s a ' o v SCHOYFELD • Department of Internal Medicine, Washington

University School of Medicine, St. Louis, MO JEFF SEEMAN • Department of Biochemistry, University of Nevada, Reno, NV RA] AjIT K. SRIVASTAVA • Department of Internal Medicine, Washington

University School of Medicine, St. Louis, MO

Contributors xiii GIORGIO STANTA • International Centre of Genetic Engineering

and Biotechnology-Padriciano (Trieste), Department of Pathology, University of Trieste

ULRIK~ STEIN • Department of Molecular and Tumor Therapy, Max-Delbruck- Centre for Molecular Medicine, Berlin, Germany

ANU SUOMALAINEN • National Public Health Institute, Department of Human Molecular Genetics, Helsinki, Finland

ANN-CHRISTINE SYVANEN • National Public Health Institute, Department of Human Molecular Genetics, Helsinki, Finland

GIORGIO TERENGHI • Blond Mclndoe Centre, Queen Victoria Hospital, Sussex, UK

BIMAL D . M . THEOPHILUS * Department of Hematology, Birmingham Children's Hospital, Birmingham, UK

WOLFGANG UCKERT • Department of Molecular and Tumor Therapy, Max-Delbruck-Centre for Molecular Medicine, Berlin, Germany

STEPHANE VIVILLE • DOI/LGME Faculte de Medicine, Strasbourg, Cedex, France MAGGIE WALMSLEY * Developmental Biology Research Centre, Kings College,

University of London, UK WOLFGANG WALTHER • Department of Oncology/Surgery, Max-Delbruck-

Centre for Molecular Medicine, Berlin, Germany FEI YE • Department of Biology, Massachusetts Institute of Technology,

Cambridge, MA