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  • Review Article
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High-throughput RNAi screening in cultured cells: a user's guide

Nature Reviews Genetics volume 7, pages 373–384 (2006)Cite this article

Key Points

  • The high-throughput, genome-scale application of RNA interference to cell-based screens — mainly in Drosophila melanogaster and mammalian cells — is revolutionizing the field of functional genomics, providing a powerful method for perturbing gene activities in cultured cells.

  • As well as direct loss-of-function screening, modifier screens can be readily conducted using RNAi, making this technique particularly useful for the analysis of signal transduction pathways.

  • RNAi has also become a method of choice for key steps in the development of therapeutic agents, from target discovery and validation to the analysis of the mechanisms of action of drug candidates, particularly through the use of modifier screens.

  • Successful implementation of a high-throughput, cell-based RNAi screen requires sophisticated infrastructure and know-how in assay design. Choosing the appropriate cell lines, screening reagents and read-out options are crucial to a successful screen.

  • In Drosophila melanogaster, all silencing reagents are based on long dsRNAs. In mammals, the choice of silencing reagent is between synthetic siRNA-like molecules, which produce only transient silencing, and vector-based shRNAs that can yield more sustained silencing.

  • Two screening paradigms can be used for large-scale RNAi screens. Systematic screens target individual genes and are the most broadly applicable in terms of the phenotypes that can be studied, but raise issues of cost. Selection-based screens use pooled libraries of shRNAs and are faster, logistically simpler and less expensive; however, a selectable phenotype is required for this type of screen.

  • Several important issues surround optimization and quality control in RNAi screens, including controlling RNAi specificity to avoid false positives, optimizing silencing to minimize false negatives, and ensuring the maximum robustness and sensitivity of the screen.

Abstract

RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.

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Figure 1: Silencing reagents for RNAi screens in mammalian cells.
Figure 2: Choosing the screening paradigm, experimental format and reagents in RNAi screens.

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Acknowledgements

We thank B. Mathey-Prevot, A. Friedman, S. Elledge, R. Zhou, K. Echeverri, C. Sachse and M. Mirotsou for helpful discussions and critical reading of the manuscript. N.P. is an investigator of the Howard Hughes Medical Institute, USA.

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Authors and Affiliations

  1. Cenix BioScience GmbH, Tatzberg 47, Dresden, 01307, Germany

    Christophe J. Echeverri

  2. Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Norbert Perrimon

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FURTHER INFORMATION

Ambion

Berkeley Drosophila Genome Project

CellProfiler cell image analysis software

Cenix Bioscience

Cytopulse

Definiens Cellenger

Dharmacon RNA Technologies

Drosophila Genome Resource Center

Drosophila RNAi Screening Center

Genetix

Invitrogen

Matrix Technologies Corporation

Open Biosystems

Panomics homepage

Qiagen homepage

Sigma-Aldrich homepage

Spotfire — Interactive Visual Analysis

TekCel

The Eurogentec web site

The MitoCheck project homepage

The RNAi Consortium

Glossary

Ribozyme

An RNA molecule with catalytic activity.

RNAi

RNA interference refers to the process by which dsRNA molecules silence a target gene through the specific destruction of its mRNA.

dsRNA

Long dsRNAs (usually referring, in this context, to those that are >200 bp in length) that are made from cDNA or genomic DNA templates.

Interferon response

A primitive antiviral mechanism that triggers sequence-nonspecific degradation of mRNA and downregulation of cellular protein synthesis.

Small interfering RNA

Small RNAs of 21–23 nucleotides in length that engage the complementary mRNA into the RISC complex for degradation.

Short hairpin RNA

Small dsRNA constructs that are usually 22–29 nucleotides long and form a hairpin-like secondary structure.

miRNA

Endogenously expressed small dsRNA (21–24 nucleotides), which can either interfere with translation of partially complementary mRNAs (usually through their 3′ end UTRs) or cause small interfering RNA-like degradation of perfectly complementary mRNAs.

Dicer

Refers to members of a highly conserved family of RNase III endonucleases that mediate dsRNA cleavage. This produces the small interfering RNAs or mature miRNAs that direct target silencing in RNAi and miRNA pathways, respectively.

Off-target effect

Any detectible phenotypic change that is triggered by RNAi treatment, other than those that are derived directly or indirectly from silencing the targeted mRNA.

High-content screens

Screens that apply multi-parametric read-outs, that is, that measure multiple phenotypic features simultaneously, usually by microscopy.

Quantitative reverse transcriptase PCR

This reaction is a sensitive method that is used to detect mRNAs.

Branched DNA assay

A signal-amplification technique that detects the presence of specific nucleic acids by measuring the signal that is generated by many branched, labelled DNA probes.

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Echeverri, C., Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nat Rev Genet 7, 373–384 (2006). https://doi.org/10.1038/nrg1836

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