Basic Science Techniques in Clinical Practice (eBook)

Foreword 5
Preface 7
Contents 9
Contributors 11
Research Governance 15
INTRODUCTION 15
WHY WE NEED RESEARCH GOVERNANCE 16
HISTORY OF DEVELOPMENT OF RESEARCH GOVERNANCE1,2 16
SALIENT FEATURES OF THE RESEARCH GOVERNANCE FRAMEWORK 19
CONCLUSION 20
References 20
Designing Health Studies 22
ESSENTIALS OF A STUDY DESIGN Specifying the Research Question 22
Selection of Subjects 22
Specifying the Primary Outcome 22
Inclusion of a Control Group 23
Confounding 23
Sample Size 24
Bias 24
Writing a Protocol 25
TWO TYPES OF STUDIES 25
Randomized Controlled Trials 25
Types of Trials 26
Observational Studies 27
References 30
Immunohistochemistry 32
INTRODUCTION 32
BASIC IMMUNOHISTOCHEMISTRY 32
METHODOLOGY 34
Fixation 35
Antigen Retrieval 35
Antibodies 36
Staining Methods/Detection Systems 37
Enzyme Labels and Chromogens 40
Animal Tissue 41
TROUBLESHOOTING/OPTIMIZATION Optimizing Primary Antibodies 41
Endogenous Enzyme Activity 42
Endogenous Biotin 43
Washing 43
References 43
Cell Culturing: A Beginner’s Guide to Understanding the Basics of Cell Culturing 45
INTRODUCTION 45
Primary Cells 45
Cell Lines 45
FUNDAMENTALS OF CELL CULTURING 46
What Happens When an Infection Occurs? 46
Media: Their Function in Cell Culturing 47
Support Cells 48
Incubation 49
CELL EXTRACTION 49
Handling Tissue Biopsies 49
Fibroblast Extraction 49
CONCLUSION 51
References 51
Flow Cytometry 52
WHAT IS FLOW CYTOMETRY? 52
HISTORICAL ASPECTS OF FLOW CYTOMETRY AND THE FLOW CYTOMETER 53
FLUORESCENCE 53
LIGHT SCATTER 55
COMPONENTS OF THE MODERN FLOW CYTOMETER Fluidic System 55
Optical System and Analysis 55
Color Assignment 56
DATA ANALYSIS Histograms and Dot Plots 57
Gating and Negative Controls 58
PRINCIPLES OF FLOW CYTOMETRY SAMPLE PREPARATION 60
References 60
Western, Northern, and Southern Blotting 62
INTRODUCTION: THE HISTORY OF SOUTHERN, NORTHERN, AND WESTERN BLOTTING 62
BASIC PRINCIPLES AND METHODS: ELECTROPHORESIS, GEL BLOTTING, AND DETECTION 63
Gel Electrophoresis: Size Separation 63
Sample Preparation: Southern and Northern 63
Sample Preparation: Western 63
Agarose Electrophoresis: Southern and Northern 64
Sodium Sodecylsulfate–Polyacrylamide Gel Electrophoresis: Western 65
Blotting: Transfer to Solid Support 66
Detection and Localization: Complementarity and Hybridization 66
Blocking 68
Hybridization 68
Washing 68
Detection 68
Stripping 70
KEY POINTS 70
References 70
Fluorescent In Situ Hybridization 72
INTRODUCTION 72
BASIC PRINCIPLES 72
METHODOLOGICAL ASPECTS OF FISH Probe and Sample Preparation 73
DNA UNMASKING: PRETREATMENT AND DIGESTION De- waxing and Rehydration 74
Acid Permeabilization 74
Reducing Agents 74
Protease Digestion 74
Probe and Target DNA Denaturation 75
Probe and Target DNA Hybridization 75
POSTHYBRIDIZATION WASH AND VISUALIZATION Posthybridization Wash 76
Visualization and Scoring/Interpretation of Results 76
QUALITY ASSURANCE 76
Slide Pretreatment 76
Acid Permeabilization 77
Pretreatment (Reducing Agent) 77
Protease Digestion 77
Denaturation 78
Hybridization 78
Posthybridization Wash 78
Visualization and Scoring Slides 79
References 79
Quantitative Reverse Transcriptase Polymerase Chain Reaction 80
INTRODUCTION 80
UNDERSTANDING THE FUNDAMENTALS 81
DISCUSSION OF METHODS TO QUANTIFY REAL-TIME POLYMERASE CHAIN REACTION RESULTS 84
CONTROLS IN QUANTITATIVE REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION: USE OF THE HOUSEKEEPING” GENE 86
AMPLICON DETECTION STRATEGIES 86
DESIGNING A PRIMER, PROBE, AND AMPLICON 90
PUSHING THE TECHNOLOGY FURTHER: MULTIPLEX QUANTITATIVE REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION 91
EQUIPMENT REVIEW 91
CONCLUSION 93
References 94
Proteonomics: High-Throughput Structural Biology — Methods for Cloning, Protein Expression, and Purification 100
INTRODUCTION 100
CURRENT APPROACHES HTP Cloning 101
Expression Systems 103
Expression Conditions 104
Expression and Solubility Screening 105
Puri.cation 105
ADDITIONAL APPROACHES 106
References 109
DNA and Tissue Microarrays 112
DNA MICROARRAY Methodology 112
Data Analysis 114
Sample Preparation Issues 115
Clinical Applications 115
TISSUE MICROARRAY Construction 116
Advantages and Criticisms of Tissue Microarrays 117
Applications 118
Quantitative Analysis 119
DNA and tissue microarray: key points 120
References 120
Basic Scientific Techniques in Recording Cellular Data 123
ELECTRICAL RECORDING SETUP 123
EXTRACELLULAR RECORDINGS 124
INTRACELLULAR RECORDINGS 125
MORPHOLOGY OF SINGLE CELLS BY INTRACELLULAR STAINING 126
OPTICAL RECORDINGS 127
CALCIUM-SENSITIVE OPTICAL IMAGING 127
VOLTAGE SENSITIVE RECORDINGS 128
INTRINSIC SIGNAL IMAGING 130
References 130
Presenting and Publishing Research Data 131
INTRODUCTION 131
OUTLETS FOR RESULTS 132
PUBLICATION IN PEER-REVIEWED JOURNALS 133
THE SPECTRUM OF JOURNALS 134
IMPACT FACTOR 134
WRITING THE PAPER 136
ASSESSMENT 137
FUTURE TRENDS 138
Ten key points 139
Analyzing Health Studies 140
OVERVIEW 140
TYPES OF DATA Numerical Data 140
Categorical Data 140
Data Structure 140
DESCRIBING DATA 141
Continuous Data 141
Categorical Data 142
ANALYZING DATA 142
Estimation 142
Hypothesis Testing 143
EXAMPLES 144
Two Independent Groups of Continuous Data 144
Two Paired Groups of Continuous Data 147
5.3 Two Independent Groups of Binary Data 149
Paired Binary Data 151
CONCLUSION 151
References 152
Future 153
Index 155