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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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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