Abstract
Regulating gene expression is a major task for all cellular systems. RNA production and degradation plays a critical role in this process and accurately measuring cellular mRNA levels is essential to understanding gene expression regulation. Classical biochemical assays that study gene expression rely on extracting RNAs from large populations of cells, taking them out of their native context and thereby losing spatial information as well as cell-to-cell variability. In this chapter, we describe a fluorescent in situ hybridization (FISH) technique that circumvents this problem by detecting single RNAs in single cells. The technique employs multiple single-stranded short DNA probes fluorescently labeled with organic dyes that hybridize to target RNAs in fixed cells, allowing quantification and localization of RNAs at the single-cell level and at single-molecule resolution. The protocol described here has been optimized for the yeast S. cerevisiae.
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References
Holstege FC, Jennings EG, Wyrick JJ et al (1998) Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95:717–728
Shendure J, Ji H (2008) Next-generation DNA sequencing. Nat Biotechnol 26:1135–1145
Elowitz M, Levine A, Siggia E, Swain P (2002) Stochastic gene expression in a single cell. Science 297:1183–1186
Larson DR, Singer RH, Zenklusen D (2009) A single molecule view of gene expression. Trends Cell Biol 19:630–637. doi:10.1016/j.tcb.2009.08.008
Locke J, Elowitz M (2009) Using movies to analyse gene circuit dynamics in single cells. Nat Rev Microbiol 7:383–392
Femino A, Fay F, Fogarty K, Singer R (1998) Visualization of single RNA transcripts in situ. Science 280:585–590
Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging technology. Nat Methods 8:S12–S19. doi:10.1038/nmeth.1573
Raj A, Peskin CS, Tranchina D et al (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4:e309. doi:10.1371/journal.pbio.0040309.sv002
Raj A, Rifkin SA, Andersen E, van Oudenaarden A (2010) Variability in gene expression underlies incomplete penetrance. Nature 463:913–918. doi:10.1038/nature08781
Raj A, van den Bogaard P, Rifkin SA et al (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5:877–879. doi:10.1038/nmeth.1253
Trcek T, Larson DR, Moldón A et al (2011) Single-molecule mRNA decay measurements reveal promoter-regulated mrna stability in yeast. Cell 147:1484–1497. doi:10.1016/j.cell.2011.11.051
Vargas DY, Shah K, Batish M et al (2011) Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell 147:1054–1065. doi:10.1016/j.cell.2011.10.024
Zenklusen D, Larson DR, Singer RH (2008) Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 15:1263–1271. doi:10.1038/nsmb.1514
Zenklusen D, Singer RH (2010) Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization. Methods Enzymol 470:641–659. doi:10.1016/S0076-6879(10)70026-4
Silverman SJ, Petti AA, Slavov N et al (2010) Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate. Proc Natl Acad Sci 107:6946–6951. doi:10.1073/pnas.s1002422107
Acknowledgements
We thank Marlene Oeffinger as well as members of the Zenklusen laboratory for comments and discussions on the manuscript. The laboratory of Daniel Zenklusen is supported by the Canadian Institutes of Health Research (MOP-BMB-232642), the Natural Sciences and Engineering Research Council of Canada, the Fonds de recherche Santé Québec, and the Canada Foundation for Innovation.
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Rahman, S., Zenklusen, D. (2013). Single-Molecule Resolution Fluorescent In Situ Hybridization (smFISH) in the Yeast S. cerevisiae . In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 1042. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-526-2_3
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DOI: https://doi.org/10.1007/978-1-62703-526-2_3
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