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Imaging rRNA Methylation in Bacteria by MR-FISH

  • Kristina A. Ganzinger
  • Martin R. Challand
  • James Spencer
  • David Klenerman
  • Rohan T. RanasingheEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)

Abstract

Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~104 to 107 cells to be pooled to generate sufficient material for analysis. Here we describe a method—methylation-sensitive RNA in situ hybridization (MR-FISH)—that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy. We outline step-by-step protocols for designing probes, in situ hybridization, and analysis of data using freely available code.

Key words

Fluorescence imaging FISH In situ hybridization DNA probes Bacteriology Antibiotic resistance Ribosomal RNA RNA modification Epitranscriptomic modifications RNA methylation Single-cell methods Microscopy Automated image analysis 

Notes

Acknowledgments

This work was supported by the EU Innovative Medicines Initiative, IMI (RAPP-ID project, grant agreement, no. 115153), the UK Biotechnology and Biological Sciences Research Council, BBSRC (Project Grant: BB/J017906/1), and the UK Engineering and Physical Sciences Research Council, EPRSC (Project Grant: EP/M027546/1). D.K. is supported by the Royal Society.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kristina A. Ganzinger
    • 1
  • Martin R. Challand
    • 2
  • James Spencer
    • 3
  • David Klenerman
    • 4
  • Rohan T. Ranasinghe
    • 4
    Email author
  1. 1.Department of Living MatterAMOLFAmsterdamThe Netherlands
  2. 2.School of BiochemistryUniversity of BristolBristolUK
  3. 3.School of Cellular and Molecular MedicineUniversity of BristolBristolUK
  4. 4.Department of ChemistryUniversity of CambridgeCambridgeUK

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