RT-PCR Protocols

394 Pages·2002·4.34 MB·English

by  Nicola King, Joe OConnell

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RT-PCR Protocols
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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
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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.
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RT-PCR Protocols/edited by Joseph O’Connell
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1. Polymerase chain reaction--Laboratory manuals. I. O’Connell, Joseph. II. Methods in
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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
v

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 DETECTIONAND ANALYSISOFMRNA
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
vii
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. DETECTIONAND ANALYSISOF 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. IN SITU LOCALIZATIONOFMRNA 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. DIFFERENTIALMRNA EXPRESSION
18 Amplified RNA for Gene Array Hybridizations
Valentina I. Shustova and Stephen J. Meltzer..............................227
Contents ix
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 INCDNA 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