Ganzinger Kristina A, Challand Martin R, Spencer James, Klenerman David, Ranasinghe Rohan T
Department of Living Matter, AMOLF, Amsterdam, The Netherlands.
School of Biochemistry, University of Bristol, Bristol, UK.
Methods Mol Biol. 2019;2038:89-107. doi: 10.1007/978-1-4939-9674-2_7.
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 ~10 to 10 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.
通常在纯化的细胞裂解物中使用下一代测序、凝胶电泳或质谱作为读数来监测RNA的甲基化。这些整体方法需要将来自约10⁶到10⁷个细胞的RNA汇集起来，以产生足够的材料用于分析。在这里，我们描述了一种方法——甲基化敏感RNA原位杂交（MR-FISH），该方法使用甲基化敏感杂交探针和荧光显微镜，在逐个细胞的基础上检测细菌中的rRNA甲基化。我们概述了使用免费可用代码设计探针、原位杂交和数据分析的分步方案。
