High-Throughput RNA Interference in Functional Genomics

  • M. Janitz
  • D. Vanhecke
  • H. Lehrach
Part of the Handbook of Experimental Pharmacology book series (HEP, volume 173)


RNA interference (RNAi) refers to post-transcriptional silencing of gene expression as a result of the introduction of double-stranded RNA into cells. The application of RNAi in experimental systems has significantly accelerated elucidation of gene functions. In order to facilitate large-scale functional genomics studies using RNAi, several high-throughput approaches have been developed based on microarray or microwell assays. The recent establishment of large libraries of RNAi reagents combined with a variety of detection assays has further improved the performance of functional genome-wide screens in mammalian cells.


RNA interference Microarray Reverse transfection Cell array 


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  1. Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) Genomewide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421:268–272CrossRefPubMedGoogle Scholar
  2. Baghdoyan S, Roupioz Y, Pitaval A, Castel D, Khomyakova E, Papine A, Soussaline F, Gidrol X (2004) Quantitative analysis of highly parallel transfection in cell microarrays. Nucleic Acids Res 32:e77CrossRefPubMedGoogle Scholar
  3. Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, Kerkhoven RM, Madiredjo M, Nijkamp W, Weigelt B, Agami R, Ge W, Cavet G, Linsley PS, Beijersbergen RL, Bernards R (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428:431–437CrossRefPubMedGoogle Scholar
  4. Boer JM, Huber WK, Sultmann H, Wilmer F, von Heydebreck A, Haas S, Korn B, Gunawan B, Vente A, Fuzesi L, Vingron M, Poustka A (2001) Identification and classification of differentially expressed genes in renal cell carcinoma by expression profiling on a global human 31,500-element cDNA array. Genome Res 11:1861–1870PubMedGoogle Scholar
  5. Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553CrossRefPubMedGoogle Scholar
  6. Carpenter AE, Sabatini DM (2004) Systematic genome-wide screens of gene function. Nat Rev Genet 5:11–22CrossRefPubMedGoogle Scholar
  7. Chittur SV (2004) DNA microarrays: tools for the 21st century. Comb Chem High Throughput Screen 7:531–537PubMedGoogle Scholar
  8. Colosimo A, Goncz KK, Holmes AR, Kunzelmann K, Novelli G, Malone RW, Bennett MJ, Gruenert DC (2000) Transfer and expression of foreign genes in mammalian cells. Biotechniques 29:314–318PubMedGoogle Scholar
  9. Conrad C, Erfle H, Warnat P, Daigle N, Lorch T, Ellenberg J, Pepperkok R, Eils R (2004) Automatic identification of subcellular phenotypes on human cell arrays. Genome Res 14:1130–1136CrossRefPubMedGoogle Scholar
  10. Erfle H, Simpson JC, Bastiaens PI, Pepperkok R (2004) siRNA cell arrays for high-content screening microscopy. Biotechniques 37:454–462PubMedGoogle Scholar
  11. Grzeskowiak R, Witt H, Drungowski M, Thermann R, Hennig S, Perrot A, Osterziel KJ, Klingbiel D, Scheid S, Spang R, Lehrach H, Ruiz P (2003) Expression profiling of human idiopathic dilated cardiomyopathy. Cardiovasc Res 59:400–411CrossRefPubMedGoogle Scholar
  12. Gutjahr C, Murphy D, Lueking A, Koenig A, Janitz M, O’Brien J, Korn B, Horn S, Lehrach H, Cahill DJ (2005) Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling. Genomics 85:285–296CrossRefPubMedGoogle Scholar
  13. Kaynak B, von Heydebreck A, Mebus S, Seelow D, Hennig S, Vogel J, Sperling HP, Pregla R, Alexi-Meskishvili V, Hetzer R, Lange PE, Vingron M, Lehrach H, Sperling S (2003) Genome-wide array analysis of normal and malformed human hearts. Circulation 107:2467–2474CrossRefPubMedGoogle Scholar
  14. Kittler R, Putz G, Pelletier L, Poser I, Heninger AK, Drechsel D, Fischer S, Konstantinova I, Habermann B, Grabner H, Yaspo ML, Himmelbauer H, Korn B, Neugebauer K, Pisabarro MT, Buchholz F (2004) An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432:1036–1040CrossRefPubMedGoogle Scholar
  15. Kumar R, Conklin DS, Mittal V (2003) High-throughput selection of effective RNAi probes for gene silencing. Genome Res 13:2333–2340CrossRefPubMedGoogle Scholar
  16. Lum L, Yao S, Mozer B, Rovescalli A, Von Kessler D, Nirenberg M, Beachy PA (2003) Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299:2039–2045CrossRefPubMedGoogle Scholar
  17. Mishina YM, Wilson CJ, Bruett L, Smith JJ, Stoop-Myer C, Jong S, Amaral LP, Pedersen R, Lyman SK, Myer VE, Kreider BL, Thompson CM (2004) Multiplex GPCR assay in reverse transfection cell microarrays. J Biomol Screen 9:196–207CrossRefPubMedGoogle Scholar
  18. Mousses S, Caplen NJ, Cornelison R, Weaver D, Basik M, Hautaniemi S, Elkahloun AG, Lotufo RA, Choudary A, Dougherty ER, Suh E, Kallioniemi O (2003) RNAi microarray analysis in cultured mammalian cells. Genome Res 13:2341–2347CrossRefPubMedGoogle Scholar
  19. Paddison PJ, Hannon GJ (2003) siRNAs and shRNAs: skeleton keys to the human genome. Curr Opin Mol Ther 5:217–224PubMedGoogle Scholar
  20. Silva JM, Mizuno H, Brady A, Lucito R, Hannon GJ (2004) RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells. Proc Natl Acad Sci USA 101:6548–6552CrossRefPubMedGoogle Scholar
  21. Stoll D, Templin MF, Bachmann J, Joos TO (2005) Protein microarrays: applications and future challenges. Curr Opin Drug Discov Devel 8:239–252PubMedGoogle Scholar
  22. van de Wetering M, Oving I, Muncan V, Pon Fong MT, Brantjes H, van Leenen D, Holstege FC, Brummelkamp TR, Agami R, Clevers H (2003) Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep 4:609–615PubMedGoogle Scholar
  23. Walter G, Bussow K, Cahill D, Lueking A, Lehrach H(2000) Protein arrays for gene expression and molecular interaction screening. Curr Opin Microbiol 3:298–302CrossRefPubMedGoogle Scholar
  24. Wang JC, Walker A, Blackwell TK, Yamamoto KR (2004) The Caenorhabditis elegans ortholog of TRAP240, CeTRAP240/let-19, selectively modulates gene expression and is essential for embryogenesis. J Biol Chem 279:29270–29277PubMedGoogle Scholar
  25. Wheeler DB, Bailey SN, Guertin DA, Carpenter AE, Higgins CO, Sabatini DM (2004) RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells. Nat Methods 1:127–132PubMedGoogle Scholar
  26. Wiemann S, Bechtel S, Bannasch D, Pepperkok R, Poustka A, German cDNA Network (2003) The German cDNA network: cDNAs, functional genomics and proteomics. J Struct Funct Genomics 4:87–96CrossRefPubMedGoogle Scholar
  27. Yoshikawa T, Uchimura E, Kishi M, Funeriu DP, Miyake M, Miyake J (2004) Transfection microarray of human mesenchymal stem cells and on-chip siRNA gene knockdown. J Control Release 96:227–232CrossRefPubMedGoogle Scholar
  28. Zheng L, Liu J, Batalov S, Zhou D, Orth A, Ding S, Schultz PG (2004) An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proc Natl Acad Sci USA 101:135–140PubMedGoogle Scholar
  29. Ziauddin J, Sabatini DM (2001) Microarrays of cells expressing defined cDNAs. Nature 411:107–110CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2006

Authors and Affiliations

  • M. Janitz
    • 1
  • D. Vanhecke
    • 1
  • H. Lehrach
    • 1
  1. 1.Department Vertebrate GenomicsMax Planck Institute for Molecular GeneticsBerlinGermany

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