A panel of highly regarded molecular biologists and clinical researchers describe in detail their most novel, useful, and interesting RT-PCR applications. Here the newcomer will find readily reproducible protocols for highly sensitive detection and quantification of gene expression, the in situ localization of gene expression in tissue, and the cloning of genes, as well as for analyzing T-cell clones and the differential expression of genes. For the expert seeking to extend the usefulness of RT-PCR, there are user-friendly applications that complement the latest technological advances, including laser-capture microdissection (LCM), real-time and quantitative PCR, microarray technology, cDNA cloning, and antibody engineering.
Author(s): Nicola King, Joe O'Connell
Series: Methods in Molecular Biology
Edition: 1st
Publisher: Humana Press
Year: 2002
Language: English
Pages: 394
Cover......Page 1
Preface......Page 6
Contents......Page 8
Contributors......Page 12
PART I. INTRODUCTION......Page 16
1 RT-PCR in Biomedicine......Page 18
2 The Basics of RT-PCR......Page 34
PART II. HIGHLY SENSITIVE DETECTION AND ANALYSIS OF MRNA......Page 42
3 Using the Quantitative Competitive RT-PCR Technique......Page 44
4 Detection of mRNA Expression and Alternative Splicing......Page 74
5 Nested RT-PCR......Page 80
6 Quantitative RT-PCR......Page 98
7 Rapid Development of a Quantitative-Competitive (qc)......Page 108
8 Quantitation of Gene Expression by RT-PCR and HPLC......Page 118
9 Time-Resolved Fluorometric Detection of Cytokine mRNAs......Page 132
10 Mimic-Based RT-PCR Quantitation of Substance P mRNA......Page 144
PART IV. DETECTION AND ANALYSIS OF RNA VIRUSES......Page 164
11 Detection and Quantification of the Hepatitis C Viral Genome......Page 166
12 Semi-Quantitative Detection of Hepatitis C Virus RNA......Page 176
13 RT-PCR for the Assessment of Genetically Heterogenous......Page 186
PART V. IN SITU LOCALIZATION OF MRNA EXPRESSION......Page 204
14 In Situ Immuno-PCR......Page 206
15 RT-PCR from Laser-Capture Microdissected Samples......Page 212
16 Mycobacterium paratuberculosis Detected by Nested PCR......Page 220
17 RT-PCR-Based Approaches to Generate Probes for mRNA......Page 228
PART VI. DIFFERENTIAL MRNA EXPRESSION......Page 240
18 Amplified RNA for Gene Array Hybridizations......Page 242
19 Semi-Quantitative Determination of Differential Gene Expression......Page 252
PART VII. GENETIC ANALYSIS......Page 266
20 Detection of Single Nucleotide Polymorphisms Using......Page 268
PART VIII. RT-PCR IN IMMUNOLOGY......Page 280
21 Detection of Clonally Expanded T-Cells by RT-PCR-SSCP......Page 282
22 Generation of scFv from a Phage Display Mini-Library Derived......Page 296
23 Generation of Murine scFv Intrabodies from B-Cell Hybridomas......Page 316
24 Quantitation of mRNA Levels by RT-PCR in Cells Purified......Page 344
PART IX. RT-PCR IN ANTI-SENSE TECHNOLOGY......Page 354
25 Detection of Anti-Sense RNA Transcripts by Anti-Sense RT-PCR......Page 356
PART X. RT-PCR IN cDNA CLONING......Page 362
26 RT-PCR in cDNA Library Construction......Page 364
27 An RT-PCR-Based Protocol for the Rapid Generation of Large,......Page 378
Index......Page 390
