Target Identification and Validation in Drug Discovery

Volume 986 of the series Methods in Molecular Biology pp 89-95


High-Throughput RNAi Screening for the Identification of Novel Targets

  • Meredith C. HendersonAffiliated withClinical Translational Research Division, Translational Genomics Research Institute (TGen)
  • , David O. AzorsaAffiliated withClinical Translational Research Division, Translational Genomics Research Institute (TGen)

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Gene silencing through RNA interference has provided researchers with an effective way to study gene function. High-throughput RNA interference (HT-RNAi) screening has further permitted researchers to identify functionally relevant mediators of cellular response on a large scale. These screens have greatly expedited the discovery of novel targets and pathway mediators. Here, we describe the methodology for performing HT-RNAi screening of HeLa cells transfected with short interfering RNA (siRNA) libraries in 384-well microplate format. Using this plate format, the HT-RNAi assay can be easily adapted to semi-automated or fully automated platforms. The library siRNA are introduced into the cells through reverse transfection using cationic lipids. HT-RNAi screening for modulators of cell proliferation can be accomplished using a single read out reagent. This type of RNAi screening can be used with most plate-based cellular assays and can be optimized for most cultured cells lines, thus becoming a powerful tool to identify specific gene modulators and targets for drug discovery.

Key words

High-throughput RNAi siRNA Library Assay Screening