Strategies for Improving RNAi Screening Success: Using a Ubiquitin-EGFP Assay to Identify Druggable Genes Required for Proteasome Function
RNA interference (RNAi) screens represent an effective method to identify novel therapeutic targets and to elucidate mechanisms of action or sensitivity to drugs. In spite of its immense potential, RNAi screening presents unique challenges that must be addressed to ensure success. We will describe the strategies employed to provide meaningful screening results. As a case study, we will discuss a cell-based assay using a ubiquitin-EGFP cell line to screen for genes that when silenced inhibit proteasome function and/or affect cell viability. We will describe the approaches used to ensure effective delivery of siRNA molecules and to identify robust siRNA controls. We will also provide strategies used to validate compatibility of the RNAi automation platform with the phenotype being analyzed. Finally, we will discuss the data analysis and hit identification methods commonly used along with approaches for follow-up analysis of potential hits from primary screening.