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Quantitative Super-Resolution Imaging of Small RNAs in Bacterial Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1737)

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).

Keywords

Fluorescence in situ hybridization Super-resolution microscopy Clustering analysis Small regulatory RNA 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyThe University of ChicagoChicagoUSA
  2. 2.The College of The University of ChicagoChicagoUSA
  3. 3.Department of Molecular Genetics and Cell BiologyThe University of ChicagoChicagoUSA
  4. 4.Institute for Biophysical DynamicsThe University of ChicagoChicagoUSA

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