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.
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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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Ganzinger, K.A., Challand, M.R., Spencer, J., Klenerman, D., Ranasinghe, R.T. (2019). Imaging rRNA Methylation in Bacteria by MR-FISH. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 2038. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9674-2_7
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DOI: https://doi.org/10.1007/978-1-4939-9674-2_7
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