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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.

References

  1. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998). Reports the discovery of RNAi that is mediated by long dsRNA in C. elegans.

    Article  CAS  Google Scholar 

  2. Fraser, A. G. et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330 (2000).

    Article  CAS  Google Scholar 

  3. Gönczy, P. et al. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331–336 (2000).

    Article  Google Scholar 

  4. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H. & Williams, B. R. Activation of the interferon system by short-interfering RNAs. Nature Cell Biol. 5, 834–839 (2003).

    Article  CAS  Google Scholar 

  5. Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl Acad. Sci. USA 97, 6499–6503 (2000). The first report that addition of long dsRNAs on cultured cells triggers a potent RNAi effect.

    Article  CAS  Google Scholar 

  6. Hamilton, A. J. & Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952 (1999).

    Article  CAS  Google Scholar 

  7. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001). The discovery that transfection of 21-nucleotide siRNAs cells triggers a potent RNAi effect.

    Article  CAS  Google Scholar 

  8. Carpenter, A. E. & Sabatini, D. M. Systematic genome-wide screens of gene function. Nature Rev. Genet. 5, 11–22 (2004). A comprehensive review of HT screens with a particularly useful overview of commercially available image-acquisition platforms.

    Article  CAS  Google Scholar 

  9. Kramer, R. & Cohen, D. Functional genomics to new drug targets. Nature Rev. Drug Discov. 3, 965–972 (2004).

    Article  CAS  Google Scholar 

  10. Kiger, A. A. et al. A functional genomic analysis of cell morphology using RNA interference. J. Biol. 2, 27 (2003).

    Article  CAS  Google Scholar 

  11. Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835 (2004).

    Article  CAS  Google Scholar 

  12. Bettencourt-Dias, M. et al. Genome-wide survey of protein kinases required for cell cycle progression. Nature 432, 980–987 (2004).

    Article  CAS  Google Scholar 

  13. Eggert, U. S. et al. Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. PLoS Biol. 2, e379 (2004). An example of how a genome-wide RNAi screen can allow drug-target identification.

    Article  Google Scholar 

  14. Lum, L. et al. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045 (2003).

    Article  CAS  Google Scholar 

  15. Nybakken, K., Vokes, S. A., Lin, T. Y., McMahon, A. P. & Perrimon, N. A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. Nature Genet. 37, 1323–1332 (2005).

    Article  CAS  Google Scholar 

  16. Baeg, G. H., Zhou, R. & Perrimon, N. Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila. Genes Dev. 19, 1861–1870 (2005).

    Article  CAS  Google Scholar 

  17. Muller, P., Kuttenkeuler, D., Gesellchen, V., Zeidler, M. P. & Boutros, M. Identification of JAK/STAT signalling components by genome-wide RNA interference. Nature 436, 871–875 (2005).

    Article  Google Scholar 

  18. Foley, E. & O'Farrell, P. H. Functional dissection of an innate immune response by a genome-wide RNAi screen. PLoS Biol. 2, e203 (2004).

    Article  Google Scholar 

  19. Gesellchen, V., Kuttenkeuler, D., Steckel, M., Pelte, N. & Boutros, M. An RNA interference screen identifies inhibitor of apoptosis protein 2 as a regulator of innate immune signalling in Drosophila. EMBO Rep. 6, 979–984 (2005).

    Article  CAS  Google Scholar 

  20. DasGupta, R., Kaykas, A., Moon, R. T. & Perrimon, N. Functional genomic analysis of the Wnt-wingless signaling pathway. Science 308, 826–833 (2005). An example of a genome-wide RNAi screen in D. melanogaster cells using a transcriptional reporter assay.

    Article  CAS  Google Scholar 

  21. Cherry, S. et al. Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition. Genes Dev. 19, 445–452 (2005).

    Article  CAS  Google Scholar 

  22. Philips, J. A., Rubin, E. J. & Perrimon, N. Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309, 1251–1253 (2005).

    Article  CAS  Google Scholar 

  23. Agaisse, H. et al. Genome-wide RNAi screen for host factors required for intracellular bacterial infection. Science 309, 1248–1251 (2005).

    Article  CAS  Google Scholar 

  24. Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004).

    Article  CAS  Google Scholar 

  25. Paddison, P. J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004).

    Article  CAS  Google Scholar 

  26. Kolfschoten, I. G. et al. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121, 849–858 (2005).

    Article  CAS  Google Scholar 

  27. Westbrook, T. F. et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848 (2005).

    Article  CAS  Google Scholar 

  28. Huesken, D. et al. Design of a genome-wide siRNA library using an artificial neural network. Nature Biotechnol. 23, 995–1001 (2005).

    Article  CAS  Google Scholar 

  29. Rottmann, S., Wang, Y., Nasoff, M., Deveraux, Q. L. & Quon, K. C. A TRAIL receptor-dependent synthetic lethal relationship between MYC activation and GSK3β/FBW7 loss of function. Proc. Natl Acad. Sci. USA 102, 15195–15200 (2005).

    Article  CAS  Google Scholar 

  30. Pelkmans, L. et al. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436, 78–86 (2005).

    Article  CAS  Google Scholar 

  31. Kittler, R. et al. An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036–1040 (2004).

    Article  CAS  Google Scholar 

  32. Nicke, B. et al. Involvement of MINK, a Ste20 family kinase, in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells. Mol. Cell 20, 673–685 (2005).

    Article  CAS  Google Scholar 

  33. Echalier, G. Drosophila Cell in Culture (Academic Press, New York, 1997).

    Google Scholar 

  34. Armknecht, S. et al. High-throughput RNA interference screens in Drosophila tissue culture cells. Methods Enzymol. 392, 55–73 (2005).

    Article  CAS  Google Scholar 

  35. Ui, K. et al. Newly established cell lines from Drosophila larval CNS express neural specific characteristics. In Vitro Cell. Dev. Biol. Anim. 30A, 209–216 (1994).

    Article  CAS  Google Scholar 

  36. Song, E. et al. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J. Virol. 77, 7174–7181 (2003).

    Article  CAS  Google Scholar 

  37. Liu, G. & Chen, X. The ferredoxin reductase gene is regulated by the p53 family and sensitizes cells to oxidative stress-induced apoptosis. Oncogene 21, 7195–7204 (2002).

    Article  CAS  Google Scholar 

  38. Ovcharenko, D., Jarvis, R., Hunicke-Smith, S., Kelnar, K. & Brown, D. High-throughput RNAi screening in vitro: from cell lines to primary cells. RNA 11, 985–993 (2005).

    Article  CAS  Google Scholar 

  39. Reynolds, A. et al. Rational siRNA design for RNA interference. Nature Biotechnol. 22, 326–330 (2004).

    Article  CAS  Google Scholar 

  40. Huang, F., Khvorova, A., Marshall, W. & Sorkin, A. Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J. Biol. Chem. 279, 16657–16661 (2004).

    Article  CAS  Google Scholar 

  41. McManus, M. T. et al. Small interfering RNA-mediated gene silencing in T lymphocytes. J. Immunol. 169, 5754–5760 (2002).

    Article  CAS  Google Scholar 

  42. Yang, D. et al. Short RNA duplexes produced by hydrolysis with Escherichia coli Rnase III mediate effective RNA interference in mammalian cells. Proc. Natl Acad. Sci. USA 99, 9942–9947 (2002).

    Article  CAS  Google Scholar 

  43. Silva, J. M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nature Genet. 37, 1281–1288 (2005).

    Article  CAS  Google Scholar 

  44. Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J. & Elledge, S. J. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc. Natl Acad. Sci. USA 102, 13212–13217 (2005).

    Article  CAS  Google Scholar 

  45. Dickins, R. A. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nature Genet. 37, 1289–1295 (2005).

    Article  CAS  Google Scholar 

  46. Ziauddin, J. & Sabatini, D. M. Microarrays of cells expressing defined cDNAs. Nature 411, 107–110 (2001).

    Article  CAS  Google Scholar 

  47. Mousses, S. et al. RNAi microarray analysis in cultured mammalian cells. Genome Res. 13, 2341–2347 (2003).

    Article  CAS  Google Scholar 

  48. Erfle, H., Simpson, J. C., Bastiaens, P. I. & Pepperkok, R. siRNA cell arrays for high-content screening microscopy. Biotechniques 37, 454–458, 460, 462 (2004).

    Article  CAS  Google Scholar 

  49. Silva, J. M., Mizuno, H., Brady, A., Lucito, R. & Hannon, G. J. RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells. Proc. Natl Acad. Sci. USA 101, 6548–6552 (2004).

    Article  CAS  Google Scholar 

  50. Wheeler, D. B. et al. RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells. Nature Methods 1, 127–132 (2004).

    Article  CAS  Google Scholar 

  51. Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21, 635–637 (2003).

    Article  CAS  Google Scholar 

  52. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods 3, 199–204 (2006).

    Article  CAS  Google Scholar 

  53. Doench, J. G., Petersen, C. P. & Sharp, P. A. siRNAs can function as miRNAs. Genes Dev. 17, 438–442 (2003).

    Article  CAS  Google Scholar 

  54. Scacheri, P. C. et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl Acad. Sci. USA 101, 1892–1897 (2004).

    Article  CAS  Google Scholar 

  55. Marques, J. T. & Williams, B. R. Activation of the mammalian immune system by siRNAs. Nature Biotechnol. 23, 1399–1405 (2005).

    Article  CAS  Google Scholar 

  56. Zhang, J. H., Chung, T. D. & Oldenburg, K. R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4, 67–73 (1999).

    Article  CAS  Google Scholar 

  57. Kim, D. H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nature Biotechnol. 23, 222–226 (2005).

    Article  CAS  Google Scholar 

  58. Bailey, S. N., Ali, S. M., Carpenter, A. E., Higgins, C. O. & Sabatini, D. M. Microarrays of lentiviruses for gene function screens in immortalized and primary cells. Nature Methods 3, 117–122 (2006).

    Article  CAS  Google Scholar 

  59. Sonnichsen, B. et al. Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434, 462–469 (2005).

    Article  CAS  Google Scholar 

  60. Ohrt, T., Merkle, D., Birkenfeld, K., Echeverri, C & Schwille, P. In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by exportin 5. Nucleic Acid Res. 34, 1369–1380 (2006).

    Article  CAS  Google Scholar 

  61. Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. & Hannon, G. J. Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235 (2004).

    Article  CAS  Google Scholar 

  62. Gregory, R. I. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).

    Article  CAS  Google Scholar 

  63. Landthaler, M., Yalcin, A. & Tuschl, T. The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14, 2162–2167 (2004).

    Article  CAS  Google Scholar 

  64. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).

    Article  CAS  Google Scholar 

  65. Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95–98 (2004).

    Article  CAS  Google Scholar 

  66. Bohnsack, M. T., Czaplinski, K. & Gorlich, D. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10, 185–191 (2004).

    Article  CAS  Google Scholar 

  67. Chendrimada, T. P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005).

    Article  CAS  Google Scholar 

  68. Saito, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Processing of pre-microRNAs by the Dicer-1–Loquacious complex in Drosophila cells. PLoS Biol. 3, e235 (2005).

    Article  Google Scholar 

  69. Forstemann, K. et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3, e236 (2005).

    Article  Google Scholar 

  70. Pillai, R. S. et al. Inhibition of translational initiation by Let-7 microRNA in human cells. Science 309, 1573–1576 (2005).

    Article  CAS  Google Scholar 

  71. Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005).

    Article  CAS  Google Scholar 

  72. MacKeigan, J. P., Murphy, L. O. & Blenis, J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nature Cell Biol. 7, 591–600 (2005).

    Article  CAS  Google Scholar 

  73. Ivanov, A. I. et al. Genes required for Drosophila nervous system development identified by RNA interference. Proc. Natl Acad. Sci. USA 101, 16216–16221 (2004).

    Article  CAS  Google Scholar 

  74. Schneider, I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27, 353–365 (1972).

    CAS  PubMed  Google Scholar 

  75. Yanagawa, S., Lee, J. S. & Ishimoto, A. Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling. J. Biol. Chem. 273, 32353–32539 (1998).

    Article  CAS  Google Scholar 

  76. Echalier, G. & Ohanessian, A. In vitro culture of Drosophila melanogaster embryonic cells. In Vitro 6, 162–172 (1970).

    Article  CAS  Google Scholar 

  77. Peel, D. J., Johnson, S. A. & Milner, M. J. The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines. Tissue Cell 22, 749–758 (1990).

    Article  CAS  Google Scholar 

  78. Kittler, R. et al. RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. Proc. Natl Acad. Sci. USA 102, 2396–2401 (2005).

    Article  CAS  Google Scholar 

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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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