Genomic screening with RNAi: results and challenges

Abstract

RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.

Figure 1

Approaches to high-throughput cell-based RNAi screening. (a) Pooled RNAi high-throughput screen (HTS) approach. (b) Arrayed RNAi HTS approach. (c) Modification of a pooled or arrayed approach via prior addition of a treatment, such as RNAi against a single gene in all cells or treatment with a small molecule. (d) Identification of related genes via informatics-based analysis (e.g., all kinases or genes previously implicated in a specific pathway or complex), followed by screening with reagents directed against the identified subset of genes. (e) A HTS with one assay type using multiple cell lines, the same cell line with multiple pathogens, or a similar multiplexed approach, followed by data integration to identify specific and general factors. (f) Parallel RNAi HTSs and an additional experimental high-throughput genomic or proteomic approach, followed by data integration to identify high-confidence hits. Abbreviations: PPI, protein-protein interaction; + sign, positive result.