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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

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High-throughput RNAi screening to dissect cellular pathways: a how-to guide.

Christina Falschlehner¹, Sandra Steinbrink, Gerrit Erdmann

¹German Cancer Research Center and University of Heidelberg, Division Signaling and Functional Genomics, Heidelberg, Germany.

Biotechnology Journal

|March 30, 2010

Summary

High-throughput RNA interference (RNAi) screens are powerful for discovering gene functions and cellular pathways. This review covers RNAi screening design, application, data analysis, and validation for reliable results.

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Area of Science:

Molecular Biology
Genetics
Cell Biology

Background:

RNA interference (RNAi) is a key technique for gene function analysis.
Genome-wide RNAi libraries facilitate high-throughput screening in diverse cell types.
RNAi screens identify novel gene functions and combined loss-of-function phenotypes.

Purpose of the Study:

To provide an overview of high-throughput RNAi screening.
To discuss the design and application of RNAi screens.
To outline data analysis and candidate validation strategies.

Main Methods:

Review of current literature on RNAi screening methodologies.
Discussion of quantitative assay design for RNAi screens.
Exploration of biological context selection for RNAi experiments.

Main Results:

High-throughput RNAi screens complement classical genetic approaches.
Successful identification of previously unknown cellular pathway components.
Assessment of complex, multifactorial genetic interactions is enabled.

Conclusions:

Effective RNAi screening relies on robust quantitative assays and appropriate biological context.
This review offers a guide for designing, implementing, and analyzing RNAi screens.
Strategies for validating candidate genes identified through RNAi screening are presented.