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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Publisher Springer Nature
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Journal Methods Mol. Biol.
Ganzinger, K.A, Challand, M, Spencer, J, Klenerman, D, & Ranasinghe, R.T. (2019). Imaging rRNA methylation in bacteria by MR-FISH. Methods Mol. Biol., 2038, 89–107. doi:10.1007/978-1-4939-9674-2_7