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Frontiers in Molecular Neuroscience (1)
PLoS ONE (1)
Gilbert, Daniel F. (2)
Abdelhay, Eliana Saul Furquim Werneck (1)
Boutros, Michael (1)
Demir, Kubilay (1)
Erdmann, Gerrit (1)
Fritzsche, Anja (1)
Islam, Robiul (1)
Jaedicke, Andreas (1)
Lynagh, Timothy (1)
Lynch, Joseph W. (1)
Muehlenberg, Katja (1)
Wanker, Erich E. (1)
Webb, Timothy I. (1)
Zhang, Xian (1)
Year of Publication
A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening
Wanker, Erich E.
Abdelhay, Eliana Saul Furquim Werneck
Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.
High Throughput Techniques for Discovering New Glycine Receptor Modulators and their Binding Sites
Lynch, Joseph W.
Webb, Timothy I.
Frontiers in Molecular Neuroscience
The inhibitory glycine receptor (GlyR) is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific modulators. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384-well plate using nanogram quantities of plasmid DNA.
Cys-loop receptor; chloride channel; pharmacology; high throughput; drug discovery; transfection
Results 1-2 (2)
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