Abstract
High-throughput RNA interference (HT-RNAi) is a powerful research tool for parallel, ‘genome-wide’, targeted knockdown of specific gene products. Such perturbation of gene product expression allows for the systematic query of gene function. The phenotypic results can be monitored by assaying for specific alterations in molecular and cellular endpoints, such as promoter activation, cell proliferation and survival. RNAi profiling may also be coupled with drug screening to identify molecular correlates of drug response. As with other genomic-scale data, methods of data analysis are required to handle the unique aspects of data normalization and statistical processing. In addition, novel techniques or knowledge-mining strategies are required to extract useful biological information from HT-RNAi data. Knowledge-mining strategies involve the novel application of bioinformatic tools and expert curation to provide biological context to genomic-scale data such as that generated from HT-RNAi data. Pathway-based tools, whether text-mining based or manually curated, serve an essential role in knowledge mining. These tools can be applied during all steps of HT-RNAi screen experiments including pre-screen knowledge gathering, assay development and hit confirmation and validation. Most importantly, pathway tools allow the interrogation of HT-RNAi data to identify and prioritize pathway-based biological information as a result of specific loss of gene function.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811.
Kennerdell, J.R. and Carthew, R.W. (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell, 95, 1017–1026.
Misquitta, L. and Paterson, B.M. (1999) Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci U S A, 96, 1451–1456.
Caplen, N.J., Parrish, S., Imani, F., Fire, A. and Morgan, R.A. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A, 98, 9742–9747.
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.
Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev, 15, 188–200.
Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. and Hannon, G.J. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science, 293, 1146–1150.
McManus, M.T. and Sharp, P.A. (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 3, 737–747.
Aza-Blanc, P., Cooper, C.L., Wagner, K., Batalov, S., Deveraux, Q.L. and Cooke, M.P. (2003) Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol Cell, 12, 627–637.
Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, M., Kerkhoven, R.M., Madiredjo, M., Nijkamp, W., Weigelt, B. et al. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature, 428, 431–437.
Brummelkamp, T.R., Fabius, A.W., Mullenders, J., Madiredjo, M., Velds, A., Kerkhoven, R.M., Bernards, R. and Beijersbergen, R.L. (2006) An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nat Chem Biol, 2, 202–206.
Chanda, S.K., White, S., Orth, A.P., Reisdorph, R., Miraglia, L., Thomas, R.S., DeJesus, P., Mason, D.E., Huang, Q., Vega, R. et al. (2003) Genome-scale functional profiling of the mammalian AP-1 signaling pathway. Proc Natl Acad Sci U S A, 100, 12153–12158.
Paddison, P.J., Silva, J.M., Conklin, D.S., Schlabach, M., Li, M., Aruleba, S., Balija, V., O’Shaughnessy, A., Gnoj, L., Scobie, K. et al. (2004) A resource for large-scale RNA-interference-based screens in mammals. Nature, 428, 427–431.
Rines, D.R., Gomez-Ferreria, M.A., Zhou, Y., Dejesus, P., Grob, S., Batalov, S., Labow, M., Huesken, D., Mickanin, C., Hall, J. et al. (2008) Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells. Genome Biol, 9, R44.
Brummelkamp, T.R., Berns, K., Hijmans, E.M., Mullenders, J., Fabius, A., Heimerikx, M., Velds, A., Kerkhoven, R.M., Madiredjo, M., Bernards, R. et al. (2004) Functional identification of cancer-relevant genes through large-scale RNA interference screens in mammalian cells. Cold Spring Harb Symp Quant Biol, 69, 439–445.
Brummelkamp, T.R., Nijman, S.M., Dirac, A.M. and Bernards, R. (2003) Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature, 424, 797–801.
MacKeigan, J.P., Murphy, L.O. and Blenis, J. (2005) Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nat Cell Biol, 7, 591–600.
Schlabach, M.R., Luo, J., Solimini, N.L., Hu, G., Xu, Q., Li, M.Z., Zhao, Z., Smogorzewska, A., Sowa, M.E., Ang, X.L. et al. (2008) Cancer proliferation gene discovery through functional genomics. Science, 319, 620–624.
Silva, J.M., Marran, K., Parker, J.S., Silva, J., Golding, M., Schlabach, M.R., Elledge, S.J., Hannon, G.J. and Chang, K. (2008) Profiling essential genes in human mammary cells by multiplex RNAi screening. Science, 319, 617–620.
Iorns, E., Lord, C.J., Turner, N. and Ashworth, A. (2007) Utilizing RNA interference to enhance cancer drug discovery. Nat Rev Drug Discov, 6, 556–568.
Whitehurst, A.W., Bodemann, B.O., Cardenas, J., Ferguson, D., Girard, L., Peyton, M., Minna, J.D., Michnoff, C., Hao, W., Roth, M.G. et al. (2007) Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature, 446, 815–819.
Caldwell, J.S. (2007) Cancer cell-based genomic and small molecule screens. Adv Cancer Res, 96, 145–173.
Echeverri, C.J., Beachy, P.A., Baum, B., Boutros, M., Buchholz, F., Chanda, S.K., Downward, J., Ellenberg, J., Fraser, A.G., Hacohen, N. et al. (2006) Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods, 3, 777–779.
Echeverri, C.J. and Perrimon, N. (2006) High-throughput RNAi screening in cultured cells: a user’s guide. Nat Rev Genet, 7, 373–384.
Konig, R., Chiang, C.Y., Tu, B.P., Yan, S.F., DeJesus, P.D., Romero, A., Bergauer, T., Orth, A., Krueger, U., Zhou, Y. et al. (2007) A probability-based approach for the analysis of large-scale RNAi screens. Nat Methods, 4, 847–849.
Perrimon, N., Friedman, A., Mathey-Prevot, B. and Eggert, U.S. (2007) Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens. Drug Discov Today, 12, 28–33.
Draghici, S., Khatri, P., Tarca, A.L., Amin, K., Done, A., Voichita, C., Georgescu, C. and Romero, R. (2007) A systems biology approach for pathway level analysis. Genome Res, 17, 1537–1545.
Draghici, S., Sellamuthu, S. and Khatri, P. (2006) Babel’s tower revisited: a universal resource for cross-referencing across annotation databases. Bioinformatics, 22, 2934–2939.
Mount, D.W. and Pandey, R. (2005) Using bioinformatics and genome analysis for new therapeutic interventions. Mol Cancer Ther, 4, 1636–1643.
Nikolsky, Y., Ekins, S., Nikolskaya, T. and Bugrim, A. (2005) A novel method for generation of signature networks as biomarkers from complex high throughput data. Toxicol Lett, 158, 20–29.
Nikolsky, Y., Nikolskaya, T. and Bugrim, A. (2005) Biological networks and analysis of experimental data in drug discovery. Drug Discov Today, 10, 653–662.
Dunn, D.A. (2002) Mining the human “kinome”. Drug Discov Today, 7, 1121–1123.
Manning, G., Whyte, D.B., Martinez, R., Hunter, T. and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science, 298, 1912–1934.
Hopkins, A.L. and Groom, C.R. (2002) The druggable genome. Nat Rev Drug Discov, 1, 727–730.
Russ, A.P. and Lampel, S. (2005) The druggable genome: an update. Drug Discov Today, 10, 1607–1610.
Eisen, M.B., Spellman, P.T., Brown, P.O. and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A, 95, 14863–14868.
Ge, H., Liu, Z., Church, G.M. and Vidal, M. (2001) Correlation between transcriptome and interactome mapping data from Saccharomyces cerevisiae. Nat Genet, 29, 482–486.
Jansen, R., Greenbaum, D. and Gerstein, M. (2002) Relating whole-genome expression data with protein-protein interactions. Genome Res, 12, 37–46.
Kemmeren, P., van Berkum, N.L., Vilo, J., Bijma, T., Donders, R., Brazma, A. and Holstege, F.C. (2002) Protein interaction verification and functional annotation by integrated analysis of genome-scale data. Mol Cell, 9, 1133–1143.
Lee, H.K., Hsu, A.K., Sajdak, J., Qin, J. and Pavlidis, P. (2004) Coexpression analysis of human genes across many microarray data sets. Genome Res, 14, 1085–1094.
Obayashi, T., Hayashi, S., Shibaoka, M., Saeki, M., Ohta, H. and Kinoshita, K. (2008) COXPRESdb: a database of coexpressed gene networks in mammals. Nucleic Acids Res, 36, D77–D82.
Chung, N., Zhang, X.D., Kreamer, A., Locco, L., Kuan, P.F., Bartz, S., Linsley, P.S., Ferrer, M. and Strulovici, B. (2008) Median absolute deviation to improve hit selection for genome-scale RNAi screens. J Biomol Screen, 13, 149–158.
Fuchs, F. and Boutros, M. (2006) Cellular phenotyping by RNAi. Brief Funct Genomic Proteomic, 5, 52–56.
Inglese, J., Shamu, C.E. and Guy, R.K. (2007) Reporting data from high-throughput screening of small-molecule libraries. Nat Chem Biol, 3, 438–441.
Aerts, S., Lambrechts, D., Maity, S., Van Loo, P., Coessens, B., De Smet, F., Tranchevent, L.C., De Moor, B., Marynen, P., Hassan, B. et al. (2006) Gene prioritization through genomic data fusion. Nat Biotechnol, 24, 537–544.
Chen, J., Xu, H., Aronow, B.J. and Jegga, A.G. (2007) Improved human disease candidate gene prioritization using mouse phenotype. BMC Bioinformatics, 8, 392.
Friend, S.H. and Oliff, A. (1998) Emerging uses for genomic information in drug discovery. N Engl J Med, 338, 125–126.
Hartwell, L.H., Szankasi, P., Roberts, C.J., Murray, A.W. and Friend, S.H. (1997) Integrating genetic approaches into the discovery of anticancer drugs. Science, 278, 1064–1068.
Shoemaker, R.H. (2006) The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer, 6, 813–823.
Weinstein, J.N. (2006) Spotlight on molecular profiling: “integromic” analysis of the NCI-60 cancer cell lines. Mol Cancer Ther, 5, 2601–2605.
Bussey, K.J., Chin, K., Lababidi, S., Reimers, M., Reinhold, W.C., Kuo, W.L., Gwadry, F., Ajay, Kouros-Mehr, H., Fridlyand, J. et al. (2006) Integrating data on DNA copy number with gene expression levels and drug sensitivities in the NCI-60 cell line panel. Mol Cancer Ther, 5, 853–867.
Scherf, U., Ross, D.T., Waltham, M., Smith, L.H., Lee, J.K., Tanabe, L., Kohn, K.W., Reinhold, W.C., Myers, T.G., Andrews, D.T. et al. (2000) A gene expression database for the molecular pharmacology of cancer. Nat Genet, 24, 236–244.
Staunton, J.E., Slonim, D.K., Coller, H.A., Tamayo, P., Angelo, M.J., Park, J., Scherf, U., Lee, J.K., Reinhold, W.O., Weinstein, J.N. et al. (2001) Chemosensitivity prediction by transcriptional profiling. Proc Natl Acad Sci U S A, 98, 10787–10792.
Zhou, Y., Young, J.A., Santrosyan, A., Chen, K., Yan, S.F. and Winzeler, E.A. (2005) In silico gene function prediction using ontology-based pattern identification. Bioinformatics, 21, 1237–1245.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kiefer, J., Yin, H.H., Que, Q.Q., Mousses, S. (2009). High-Throughput siRNA Screening as a Method of Perturbation of Biological Systems and Identification of Targeted Pathways Coupled with Compound Screening. In: Nikolsky, Y., Bryant, J. (eds) Protein Networks and Pathway Analysis. Methods in Molecular Biology, vol 563. Humana Press. https://doi.org/10.1007/978-1-60761-175-2_15
Download citation
DOI: https://doi.org/10.1007/978-1-60761-175-2_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-174-5
Online ISBN: 978-1-60761-175-2
eBook Packages: Springer Protocols