An analysis of normalization methods for Drosophila RNAi genomic screens and development of a robust validation scheme.

Genome-wide RNA interference (RNAi) screening allows investigation of the role of individual genes in a process of choice. Most RNAi screens identify a large number of genes with a continuous gradient in the assessed phenotype. Screeners must decide whether to examine genes with the most robust phenotype or the full gradient of genes that cause an effect and how to identify candidate genes. The authors have used RNAi in Drosophila cells to examine viability in a 384-well plate format and compare 2 screens, untreated control and treatment. They compare multiple normalization methods, which take advantage of different features within the data, including quantile normalization, background subtraction, scaling, cellHTS2 (Boutros et al. 2006), and interquartile range measurement. Considering the false-positive potential that arises from RNAi technology, a robust validation method was designed for the purpose of gene selection for future investigations. In a retrospective analysis, the authors describe the use of validation data to evaluate each normalization method. Although no method worked ideally, a combination of 2 methods, background subtraction followed by quantile normalization and cellHTS2, at different thresholds, captures the most dependable and diverse candidate genes. Thresholds are suggested depending on whether a few candidate genes are desired or a more extensive systems-level analysis is sought. The normalization approaches and experimental design to perform validation experiments are likely to apply to those high-throughput screening systems attempting to identify genes for systems-level analysis.