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Single mRNA Molecule Detection in Drosophila

  • Protocol
RNA Detection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1649))

Abstract

Single molecule fluorescent in situ hybridization (smFISH) enables quantitative measurements of gene expression and mRNA localization. The technique is increasingly popular for analysis of cultured cells but is not widely applied to intact organisms. Here, we describe a method for labeling and detection of single mRNA molecules in whole embryos of the fruit fly Drosophila melanogaster. This method permits measurements of gene expression in absolute units, enabling new studies of transcriptional mechanisms underlying precision and reproducibility in cell specification.

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References

  1. Chen H, Larson DR (2016) What have single-molecule studies taught us about gene expression? Genes Dev 30(16):1796–1810. doi:10.1101/gad.281725.116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Chong S, Chen C, Ge H, Xie XS (2014) Mechanism of transcriptional bursting in bacteria. Cell 158(2):314–326. doi:10.1016/j.cell.2014.05.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gandhi SJ, Zenklusen D, Lionnet T, Singer RH (2011) Transcription of functionally related constitutive genes is not coordinated. Nat Struct Mol Biol 18(1):27–34. doi:10.1038/nsmb.1934

    Article  CAS  PubMed  Google Scholar 

  4. Little SC, Tikhonov M, Gregor T (2013) Precise developmental gene expression arises from globally stochastic transcriptional activity. Cell 154(4):789–800. doi:10.1016/j.cell.2013.07.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4(10):e309. doi:10.1371/journal.pbio.0040309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X (2015) RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348(6233):aaa6090. doi:10.1126/science.aaa6090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schwabe A, Bruggeman FJ (2014) Single yeast cells vary in transcription activity not in delay time after a metabolic shift. Nat Commun 5:4798. doi:10.1038/ncomms5798

    Article  CAS  PubMed  Google Scholar 

  8. Skinner SO, Xu H, Nagarkar-Jaiswal S, Freire PR, Zwaka TP, Golding I (2016) Single-cell analysis of transcription kinetics across the cell cycle. Elife 5:e12175. doi:10.7554/eLife.12175

    Article  PubMed  PubMed Central  Google Scholar 

  9. Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M, Hearn J, Emili A, Xie XS (2010) Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329(5991):533–538. doi:10.1126/science.1188308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bahar Halpern K, Tanami S, Landen S, Chapal M, Szlak L, Hutzler A, Nizhberg A, Itzkovitz S (2015) Bursty gene expression in the intact mammalian liver. Mol Cell 58(1):147–156. doi:10.1016/j.molcel.2015.01.027

    Article  CAS  PubMed  Google Scholar 

  11. Bayer LV, Batish M, Formel SK, Bratu DP (2015) Single-molecule RNA in situ hybridization (smFISH) and immunofluorescence (IF) in the Drosophila egg chamber. Methods Mol Biol 1328:125–136. doi:10.1007/978-1-4939-2851-4_9

    Article  CAS  PubMed  Google Scholar 

  12. Bolkova J, Lanctot C (2016) Quantitative gene expression analysis in Caenorhabditis elegans using single molecule RNA FISH. Methods 98:42–49. doi:10.1016/j.ymeth.2015.11.008

    Article  CAS  PubMed  Google Scholar 

  13. Stapel LC, Lombardot B, Broaddus C, Kainmueller D, Jug F, Myers EW, Vastenhouw NL (2016) Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos. Development 143(3):540–546. doi:10.1242/dev.128918

    Article  CAS  PubMed  Google Scholar 

  14. Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590

    Article  CAS  PubMed  Google Scholar 

  15. Little SC, Tkacik G, Kneeland TB, Wieschaus EF, Gregor T (2011) The formation of the Bicoid morphogen gradient requires protein movement from anteriorly localized mRNA. PLoS Biol 9(3):e1000596. doi:10.1371/journal.pbio.1000596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Petkova MD, Little SC, Liu F, Gregor T (2014) Maternal origins of developmental reproducibility. Curr Biol 24(11):1283–1288. doi:10.1016/j.cub.2014.04.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. doi:10.1038/nmeth.1253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bachmann A, Knust E (2008) The use of P-element transposons to generate transgenic flies. Methods Mol Biol 420:61–77. doi:10.1007/978-1-59745-583-1_4

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 1112 Biomedical Research Bldg. II/III, 421 Curie Blvd., Philadelphia, PA, 19104, USA

    Shawn C. Little

  2. Department of Physics, Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Thomas Gregor

Authors
  1. Shawn C. Little
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  2. Thomas Gregor
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Corresponding author

Correspondence to Shawn C. Little .

Editor information

Editors and Affiliations

  1. Developmental Biology Unit, EMBL Heidelberg, Heidelberg, Germany

    Imre Gaspar

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Little, S.C., Gregor, T. (2018). Single mRNA Molecule Detection in Drosophila . In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_8

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  • DOI: https://doi.org/10.1007/978-1-4939-7213-5_8

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7212-8

  • Online ISBN: 978-1-4939-7213-5

  • eBook Packages: Springer Protocols

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