High-throughput RNAi screening to dissect cellular pathways: a how-to guide
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Updated: Jun 14, 2026

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Christina Falschlehner1, Sandra Steinbrink, Gerrit Erdmann
1German Cancer Research Center and University of Heidelberg, Division Signaling and Functional Genomics, Heidelberg, Germany.
Abstract:
RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.
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