RNA Purification and Analysis Sample

This first book on the market covers the many new and important RNA species discovered over the past five years, explaining current methods for the enrichment, separation and purification of these novel RNAs. Building up from general principles of RNA biochemistry and biophysics, this book addresses the practical aspects relevant to the laboratory researcher throughout, while discussing the performance and potential problems of the methods discussed. An appendix contains a glossary with the important terms and techniques used in RNA analysis. By explaining the basic and working principles of the methods, the book allows biochemists and molecular biologists to gain much more expertise than by simply repeating a pre-formulated protocol, enabling them to select the procedure and materials best suited to the RNA analysis task at hand. As a result, they will be able to develop new protocols where needed and optimize and fine-tune the general purpose standard protocols that come with the purification equipment and instrumentation.

Douglas Gjerde received his PhD in analytical chemistry from Iowa State University (USA). After having worked in large and small companies for a number of years, Dr. Gjerde founded Sarasep (now merged into Transgenomic) in 1990 for the production and commercialization of nucleic acid separation technologies. He has authored and coauthored numerous articles and 4 books and holds nearly fifty patents in separation science.

Lee Hoang is a graduate of the University of California, Santa Cruz (USA) where he obtained a Ph.D. in Molecular Biology in 2006. He is now an applications scientist at PhyNexus in San Jose, a company specializing on protein purification that was founded by Douglas Gjerde.

David Hornby studied biochemistry at the University of Sheffield (UK). Since 1999 he is a full professor for Molecular Biology and Biotechnology at the same institution. He has also co-founded two start-up companies (Invector and Protealysis), based on novel technologies for RNA separation and stabilisation, for which he holds the patents. His current research focuses on RNAi biochemistry and proteomics technologies.

Preface	p. IX
Acknowledgments	p. XI
RNA Extraction, Separation, and Analysis	p. 1
The Need to Be Able to Extract, Manipulate, and Analyze RNA	p. 1
Using Chemical Tools to Solve the Problem of Analysis of Biological Processes	p. 3
The Principle of Chromatography and Solid-Phase Extraction	p. 4
Principle of Chromatography	p. 4
Mobile Phase Gradient Controls Elution	p. 5
Different Types of Column and Eluent Chemistries	p. 6
The Principle of Solid-Phase Extraction	p. 8
RNA Chromatography	p. 10
Enzymatic Treatment of RNA and Analysis	p. 13
Polyacrylamide Gel Electrophoresis	p. 14
RNA Structure Probing with Ribonuclease Enzymes	p. 14
Content and Organization of This Book	p. 15
References	p. 16
Biological and Chemical RNA	p. 17
Why Classify RNA with Biology and Chemistry?	p. 17
Chemical Classification of RNA	p. 18
Biological Classification of RNA	p. 19
Prokaryotic Cellular RNA	p. 20
Prokaryote Sample Type	p. 23
Escherichia coli	p. 23
Other Bacteria	p. 24
Eukaryotic Cellular RNA	p. 24
Eukaryote Sample Type	p. 27
Yeast	p. 29
Other Fungi	p. 30
Simple Multicellular Organism	p. 30
Soft Animal	p. 30
Hard Animal	p. 31
Plant	p. 31
Cell Culture	p. 31
Other Samples	p. 32
Virus	p. 32
Soil and Rock	p. 32
Synthetic RNA	p. 32
Aptamers	p. 33
SELEX	p. 34
Short Hairpin RNAs	p. 34
References	p. 34
RNA Separation: Substrates, Functional Groups, Mechanisms, and Control	p. 37
Solid-Phase Interaction	p. 37
Adsorption of Sample Compounds and Sample Matrix Compounds	p. 37
Roles of Solid-Phase Substrate and Functional Group	p. 39
Correlation of Interaction Type, Functional Group, and Substrate	p. 40
RNA Structure and Solid Surface Interaction	p. 41
The Solid-Phase Substrate and Attachment of Functional Groups	p. 43
Polymeric Resin Substrates	p. 44
Porous and Nonporous Polymeric Resins	p. 45
Monolith Polymeric Columns	p. 47
Functionalization of the Polymer	p. 48
Silica-Glass-Based Substrates	p. 50
Functionalization of Silica	p. 51
Agarose and Cellulose Affinity Substrates	p. 53
Dextran and Polyacrylamide Gel Filtration Substrates	p. 53
Reverse-Phase Ion-Pairing Separation Mechanism	p. 54
Ion-Exchange Separation Mechanism	p. 57
Chaotropic Denaturing Interaction Mechanism	p. 61
Hybridization	p. 62
SELEX	p. 62
Gel Filtration	p. 63
References	p. 64
RNA Extraction and Analysis	p. 67
Transcription	p. 67
RNA Catalysis	p. 69
RNA-Protein Complex Interactions	p. 69
Pre-mRNA Splicing	p. 71
Translation	p. 72
Post-Transcriptional Control of Eukaryotic Gene Expression	p. 76
Gene Regulation	p. 76
RNA Interference Pathway	p. 76
Micro RNAs arid Their Role in Gene Regulation	p. 78
Use of siRNA to Investigate Gene Function	p. 79
References	p. 79
RNA Chromatography	p. 81
Development of RNA Chromatography	p. 81
RNA Chromatography Instrumentation	p. 85
The Column Oven	p. 85
Ultraviolet (UV) and Fluorescence Detection	p. 86
Fragment Collection	p. 86
RNA Chromatography Conditions	p. 87
Temperature Modes of RNA Chromatography	p. 88
Nondenaturing Mode	p. 89
Partially Denaturing Mode	p. 89
Fully Denaturing Mode	p. 90
Comparison of Gel Electrophoresis and Liquid Chromatography	p. 90
Gel Electrophoresis	p. 90
Liquid Chromatography	p. 93
Analysis of Human Telomerase RNA Under Nondenaturing Conditions	p. 95
References	p. 98
RNA Chromatography Separation and Analysis	p. 101
Features of RNA Chromatography	p. 101
Separation of Double-Stranded and Single-Stranded RNA	p. 102
Separation of Cellular RNA Species	p. 107
Separation of Messenger RNA from Total and Ribosomal RNA	p. 108
Analysis of Transfer RNA	p. 109
Chromatography and Analysis of Synthetic Oligoribonucleotides	p. 111
Application of RNA and DNA Chromatography in cDNA Library Synthesis	p. 114
DNA Chromatography Analyses of RT-PCR and Competitive RT-PCR Products	p. 117
Alternative Splicing	p. 120
Differential Messenger RNA Display via DNA Chromatography	p. 121
References	p. 124
RNA Structure-Function Probing	p. 127
Definition of the Structure-Function Paradigm	p. 127
Francis Crick, and Predicting the Existence of tRNA	p. 130
The Discovery of Ribozymes	p. 133
Structure Determination of RNA	p. 135
Primary Structure	p. 136
Secondary Structure	p. 136
Tertiary Structure	p. 137
Quaternary Structure	p. 138
Footprinting, Model Building, and Functional Investigations	p. 139
Chemical Probing and Cleavage	p. 140
Modification Interference	p. 143
FRET	p. 144
References	p. 144
Chromatographic Separation Equations and Principles for RNA Separation	p. 147
HPLC Instrumentation and Operation	p. 159
RNA Chromatographic System Cleaning and Passivation Treatment	p. 185
Index	p. 191
Table of Contents provided by Ingram. All Rights Reserved.

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RNA Purification and Analysis Sample Preparation, Extraction, Chromatography

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