Strategies for Improving RNAi Screening Success: Using a Ubiquitin-EGFP Assay to Identify Druggable Genes Required for Proteasome Function
Poster Dec 15, 2016
Anja van Brabant Smith, Elena Maksimova, Matthew Brenton, Mark Deiparine, Rachel Edwards, Phoenix Kwan, Amanda Birmingham, Devin Leake
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.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE