Abstract
Bioinformatics programs have helped tremendously in identifying the targets of microRNAs, which are small noncoding RNAs that regulate gene expression posttranscriptionally. However, the partial complementarity between miRNAs and their targets hinders the accuracy of target prediction, necessitating the use of experimental validation procedures. Here, we describe a gene reporter assay typically used in our lab to validate putative miRNA–mRNA interactions in Drosophila S2 cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10:94–108
Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10:126–139
Bartel DP, Chen CZ (2004) Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat Rev Genet 5:396–400
Pauli A, Rinn JL, Schier AF (2011) Non-coding RNAs as regulators of embryogenesis. Nat Rev Genet 12:136–149
Yekta S, Tabin CJ, Bartel DP (2008) MicroRNAs in the Hox network: an apparent link to posterior prevalence. Nat Rev Genet 9:789–796
Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12:99–110
Berezikov E, Guryev V, van de Belt J et al (2005) Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120:21–24
Lai EC, Tomancak P, Williams RW et al (2003) Computational identification of Drosophila microRNA genes. Genome Biol 4:R42
Lim LP, Glasner ME, Yekta S et al (2003) Vertebrate microRNA genes. Science 299:1540
Bartel DP (2009) microRNAs: target recognition and regulatory functions. Cell 136:215–233
John B, Enright AJ, Aracin A et al (2004) Human microRNA targets. PLoS Biol 2:e363
Krek A et al (2005) Combinatorial microRNA target predictions. Nat Genet 37:495–500
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20
Orom UA, Lund AH (2009) Experimental identification of microRNA targets. Gene 451:1–5
Thomson DW, Bracken CP, Goodall GJ (2011) Experimental strategies for microRNA target identification. Nucleic Acids Res 39:6845–6853
Karginov FV, Conaco C, Xuan Z et al (2007) A biochemical approach to identifying microRNA targets. Proc Natl Acad Sci U S A 104:19291–19296
Chi SW, Zang JB, Mele A et al (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460: 479–486
Bracken CP, Gregory PA, Kolesnikoff N et al (2008) A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res 68:7846–7854
Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York
Gloor GB, Preston CR, Johnson-Schlitz DM et al (1993) Type I repressors of P element mobility. Genetics 135:81–95
Acknowledgements
This work was supported by the Scientific and Technical Research Council of Turkey (104T144 to BA). We also thank the IZTECH Center for Biotechnology for their help.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Akgül, B., Göktaş, Ç. (2014). Gene Reporter Assay to Validate MicroRNA Targets in Drosophila S2 Cells. In: Yousef, M., Allmer, J. (eds) miRNomics: MicroRNA Biology and Computational Analysis. Methods in Molecular Biology, vol 1107. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-748-8_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-748-8_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-747-1
Online ISBN: 978-1-62703-748-8
eBook Packages: Springer Protocols