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.
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.
RNA interference (RNAi) using small interfering RNAs (siRNAs) is an important technology for down-regulation of gene expression and a powerful tool to study cellular processes and pathways. Previously, large collections of siRNAs were available only for traditional experimental model systems, such as human and mouse, and predominantly provided as chemically synthesized libraries.READ MORE