Abstract
We present a method for the quantification of small regulatory RNAs (sRNAs) in bacteria, by combining single-molecule fluorescence in situ hybridization (smFISH), super-resolved single-fluorophore microscopy, and clustering analysis. Compared to smFISH imaging with diffraction-limited fluorescence microscopy, our method provides better quantification for short and abundant RNA (such as sRNAs) in a small volume of bacterial cells. Our method can also be directly used for the quantification of messenger RNAs (mRNAs).
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Acknowledgments
The data presented as examples were collected during the 2016 Center for the Physics of Living Cells (CPLC) Summer School at University of Illinois at Urbana-Champaign. We therefore thank National Science Foundation grant PHY-1430124 (Physics Frontiers Center for the Physics of Living Cells) for funding for the CPLC Summer School, Zan Luthey-Schulten and Joseph R. Peterson for co-teaching the scientific theme of “Quantitative Imaging and Cell Simulation of Small Regulatory RNA,” and the summer school students: Bijoy Desai (Columbia University), Dennis Fernandes (University of Toronto, Mississauga), Jialei Tang (University of Central Florida), and Kailun Zhang (Texas A&M University) for participation in data collection and analysis.
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Park, S., Bujnowska, M., McLean, E.L., Fei, J. (2018). Quantitative Super-Resolution Imaging of Small RNAs in Bacterial Cells. In: Arluison, V., Valverde, C. (eds) Bacterial Regulatory RNA. Methods in Molecular Biology, vol 1737. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7634-8_12
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DOI: https://doi.org/10.1007/978-1-4939-7634-8_12
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