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Counting mRNA Copies in Intact Bacterial Cells by Fluctuation Localization Imaging-Based Fluorescence In Situ Hybridization (fliFISH)

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Fluorescence In-Situ Hybridization (FISH) for Microbial Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2246))

Abstract

A method for measuring mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) is presented. Unlike conventional single-molecule FISH, where the presence of a transcript is determined by fluorescence intensity, fliFISH relies on On-Off duty cycles of photo-switching dyes to set a predetermined threshold for distinguishing true signals from background noise. The method provides a quantitative approach for detecting and counting true mRNA copies and rejecting false signals with high accuracy.

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Acknowledgments

We thank Dr. Timothy McDermott at the Land Resources & Environmental Sciences, Montana State University, for providing us with E. coli expressing AAT. The majority of the work was done at the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by DOE’s Office of Biological and Environmental Research (BER) and located at Pacific Northwest National Laboratory. Part of the work was done under the Facilities Integrating Collaborations for User Science (FICUS), using resources at EMSL and the Joint Genome Institute (JGI). Both are DOE scientific user facilities sponsored by BER and operated under Contract Nos. DE-AC05-76RL01830 (EMSL) and DE-AC02-05CH11231 (JGI).

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Author notes

  1. Yi Cui

    Present address: MIT Media Lab, Cambridge, MA, USA

Authors and Affiliations

  1. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA

    Dehong Hu, Yi Cui, Lye M. Markillie, William B. Chrisler & Galya Orr

  2. Land Resources & Environmental Sciences, Montana State University, Bozeman, MT, USA

    Qian Wang

  3. Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

    Roland Hatzenpichler

Authors
  1. Dehong Hu
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  2. Yi Cui
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  3. Lye M. Markillie
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  4. William B. Chrisler
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  5. Qian Wang
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  6. Roland Hatzenpichler
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  7. Galya Orr
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Corresponding author

Correspondence to Galya Orr .

Editor information

Editors and Affiliations

  1. LEPABE – Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, University of Porto, Porto, Portugal

    Nuno F. Azevedo

  2. INIAV – National Institute for Agrarian and Veterinarian Research, Rua dos Lagidos, Lugar da Madalena, Vairão, CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, LEPABE – Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, University of Porto, Vila do Conde, Portugal

    Carina Almeida

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Hu, D. et al. (2021). Counting mRNA Copies in Intact Bacterial Cells by Fluctuation Localization Imaging-Based Fluorescence In Situ Hybridization (fliFISH). In: Azevedo, N.F., Almeida, C. (eds) Fluorescence In-Situ Hybridization (FISH) for Microbial Cells. Methods in Molecular Biology, vol 2246. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1115-9_15

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  • DOI: https://doi.org/10.1007/978-1-0716-1115-9_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1114-2

  • Online ISBN: 978-1-0716-1115-9

  • eBook Packages: Springer Protocols

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