Book contents
- Frontmatter
- Contents
- Preface
- Acknowledgments
- Acronyms and Abbreviations
- Part I RNAi HTS and Data Analysis
- 1 Introduction to Genome-Scale RNAi Research
- 2 Experimental Designs
- 3 Data Display and Normalization
- 4 Quality Control in Genome-Scale RNAi Screens
- 5 Hit Selection in Genome-Scale RNAi Screens without Replicates
- 6 Hit Selection in Genome-Scale RNAi Screens with Replicates
- Part II Methodological Development for Analyzing RNAi HTS Screens
- References
- Index
- Plate section
5 - Hit Selection in Genome-Scale RNAi Screens without Replicates
Published online by Cambridge University Press: 03 May 2011
- Frontmatter
- Contents
- Preface
- Acknowledgments
- Acronyms and Abbreviations
- Part I RNAi HTS and Data Analysis
- 1 Introduction to Genome-Scale RNAi Research
- 2 Experimental Designs
- 3 Data Display and Normalization
- 4 Quality Control in Genome-Scale RNAi Screens
- 5 Hit Selection in Genome-Scale RNAi Screens without Replicates
- 6 Hit Selection in Genome-Scale RNAi Screens with Replicates
- Part II Methodological Development for Analyzing RNAi HTS Screens
- References
- Index
- Plate section
Summary
Introduction
In an RNAi HTS, one primary goal is to select siRNAs with a desired size of inhibition or activation effect. The size of the siRNA effect is represented by the magnitude of difference between a tested siRNA and a negative reference group with no specific inhibition/activation effects. An siRNA with a desired size of effects in an HTS screen is called a hit. The process of selecting hits is called hit selection. There are two main strategies of selecting hits with large effects. One is to use certain metric(s) to rank and/or classify the siRNAs by their effects and then to select the largest number of potent siRNAs that is practical for validation assays. The other strategy is to test whether an siRNA has effects strong enough to reach a pre-set level. In this strategy, false-negative rates (FNRs) and/or false-positive rates (FPRs) must be controlled.
As described in Chapter 1, a typical RNAi HTS project currently starts with a primary screen of single or pooled siRNAs, most of which have no replicate, and follows with one or more confirmatory screens in which each siRNA or pool has replicates. With the development of the platform of 1,536-well plates, more and more primary screens can also have replicates. The analytic methods for hit selection in screens without replicates differ from those with replicates. Therefore, I discuss these methods separately: without replicates in this chapter and with replicates in Chapter 6.
- Type
- Chapter
- Information
- Optimal High-Throughput ScreeningPractical Experimental Design and Data Analysis for Genome-Scale RNAi Research, pp. 62 - 82Publisher: Cambridge University PressPrint publication year: 2011