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RT-PCR Protocols - Joe O’Connell

Contents


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

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. IN SITU 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

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