Optimal High-Throughput Screening: Practical Experimental Design and Data Analysis for Genome-Scale RNAi Research

Half-title......Page 3
Title......Page 5
Copyright......Page 6
Contents......Page 7
Preface......Page 11
Audience......Page 13
Content and General Outline......Page 14
Acknowledgments......Page 17
Acronyms and Abbreviations......Page 19
Part I: RNAi HTS and Data Analysis......Page 21
1.1 RNAi: An Effective Tool for Elucidating Gene Functions and a New Class of Drugs......Page 23
1.2 High-Throughput Screening: A Vital Technology in Drug Discovery......Page 25
1.3 Genome-Scale RNAi Screens......Page 27
1.4 An Example of Genome-Scale RNAi Research......Page 29
1.5 Challenges in Genome-Scale RNAi Research......Page 30
2.1 siRNA Designs......Page 33
2.2.2 Design of Positive Controls......Page 35
2.3 Plate Designs......Page 36
2.3.1 Construction of Plate Designs......Page 37
2.3.2 Capacity of Plate Designs in Identifying and Adjusting Spatial Effects......Page 40
2.3.3 Guidelines for Adopting Plate Designs in HTS Experiments......Page 43
2.5 Design of Sample Size......Page 44
2.6 Conclusions......Page 45
3.1.1 Plate-Well Series Plot......Page 47
3.1.2 Plate Image Plots Incorporating Boxplot and Heat Maps......Page 48
3.2 Transformation of Measured Raw Values......Page 52
3.3.1 Identifying Systematic Spatial Effects......Page 53
3.3.2 Consequence of Unadjusted Spatial Effects......Page 54
3.3.3 Methods for Adjusting for Systematic Spatial Effects......Page 56
3.3.4 Applications in Adjustment of Systematic Spatial Effects......Page 59
3.4 Strategy for Data Display and Normalization......Page 61
4.2 Quality Assessment Metrics......Page 62
4.3 Quality Control Criteria......Page 67
4.4 Adoption of Effective Plate Designs......Page 73
4.5 Integration of Experimental and Analytic Approaches to Improve Data Quality......Page 76
4.6 Application......Page 77
4.7 Discussion and Conclusions......Page 80
5.1 Introduction......Page 82
5.2.1 A Negative Reference to Represent siRNAs with No Specific Effects......Page 83
5.2.3 Hit Selection Metrics and Their Calculation......Page 84
5.2.4 Comparison of Metrics for Hit Selection Using Example Data Sets......Page 87
5.3 Decision Rules for Hit Selection in RNAi Screens......Page 89
5.3.2 Decision Rules in the z-Score Method......Page 90
5.3.3 SSMD-Based Decision Rules......Page 91
5.4 Sample Size Determination......Page 94
5.5 Applications......Page 97
5.6 Conclusions......Page 102
6.1 Metrics for Hit Selection in Screens with Replicates......Page 103
6.1.2 SSMD Method......Page 104
6.2 Dual-Flashlight Plot......Page 106
6.3 Decision Rules for Hit Selection in Screens with Replicates......Page 108
6.4 False Discovery Rate, False Non-Discovery Rate, q-Value, and q*-Value......Page 110
6.5 Sample Size Determination......Page 112
6.6.1 Introduction to Experiments Addressing Off-Target Effects......Page 113
6.6.2 Issues in Current Analytic Methods Addressing Off-Target Effects......Page 114
6.6.3 SSMD Methods Assessing Collective Activity of Multiple siRNAs......Page 115
6.6.4 SSMD-Based Methods Assessing Off-Target Effects and Specific On-Target Effects......Page 117
6.7.1 An Example of an RNAi Confirmatory Screen with Replicates......Page 118
6.7.2 An Example of an RNAi Deconvolution Screen with Multiple Single siRNAs per Gene......Page 121
6.8 Discussion and Conclusions......Page 126
Part II: Methodological Development for Analyzing RNAi HTS Screens......Page 129
7 Statistical Methods for Group Comparison......Page 131
7.1 Illustration of Issues in Traditional Contrast Analysis......Page 132
7.2 Contrast Variable, SMCV, and c+-Probability......Page 134
7.3 A Classifying Rule for Interpreting Strength of Group Comparisons......Page 136
7.4 A Theorem to Facilitate the Estimation and Inference of SMCV......Page 139
7.5 Estimation of SMCV and c+-Probability......Page 147
7.5.1 Estimation of SMCV in Unmatched Samples......Page 148
7.5.2 Estimation of SMCV in Matched Samples......Page 151
7.6.1 A Contrast in General......Page 153
7.6.2 Effects of a Combination of Factor Levels......Page 157
7.6.3 Main Effects......Page 158
7.6.4 Interaction Effects......Page 161
7.7.1 A Simulation Study......Page 162
7.7.2 An Example of Matched Contrast Analysis......Page 165
7.7.3 An Illustrative Example for Contrast Variables in ANOVA......Page 167
7.7.4 An Illustrative Example for Phenotypic Effects of an siRNA in Multifactor ANOVA......Page 170
7.8 Discussion and Conclusions......Page 172
8.1 SSMD and d+-Probability......Page 174
8.2 Estimation of SSMD......Page 176
8.3.1 Classical t-Test and Standardized Mean Difference......Page 179
8.3.2 Comparing SSMD with Classical t-Test......Page 181
8.3.3 Comparing SSMD with Standardized Mean Difference......Page 183
8.4 SSMD-Based Ranking Methods for Hit Selection in Genome-Scale RNAi Screens......Page 185
8.5.1 FPR, FNR, and Power in RNAi Screens......Page 186
8.5.2 Classical Hypothesis Test and SSMD-Based Error Control Method......Page 187
8.5.3 Controlling FPR and FNR in Genome-Scale RNAi Screens......Page 189
8.5.4 p-Value and p*-Value in RNAi Screens......Page 192
8.5.5 Controlling Power in RNAi Screens......Page 193
8.6.1 Basic Concepts of FDR......Page 196
8.6.2 q-Value......Page 197
8.6.3 Estimation of q-Value and FDR......Page 198
8.7.1 Introduction to Off-Target Effects......Page 199
8.7.2 Model for Collective Activity of Multiple siRNAs......Page 200
8.7.3 Estimation of Mean and SSMD for the Collective Activity of Multiple siRNAs......Page 202
8.7.5 Specific Activity of an Individual siRNA......Page 203
8.8 Discussion and Conclusions......Page 206
References......Page 209
Index......Page 221