Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridisation (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridised slices of high pressure frozen, LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localisation that can be complemented with immunofluorescence and electron microscopy, as well as electron tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA tertiary structures, and we show that the method can be employed to probe for mRNA compactness. We apply LR White smFISH to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing, using trypanosomes and their versatile RNA biology as a model system.

中文翻译：

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用相关的单分子FISH对LR White包埋样品上的mRNA丰度，定位和紧密度进行并行监测

通常使用分支DNA技术通过单分子荧光原位杂交（smFISH）检测单个mRNA分子。尽管提供了强大且背景减弱的信号，但该方法在检测密集结构内的mRNA，监测mRNA的紧密性和定量丰富的mRNA方面效率低下。为克服这些限制，我们将高压冷冻的LR White嵌入式细胞（LR White smFISH）的切片进行了杂交。mRNA检测在物理上仅限于树脂表面。不管其丰度如何，它都能够以与RNA测序相当的精度检测RNA，同时提供有关RNA定位的空间信息，可以用免疫荧光和电子显微镜以及电子断层摄影术进行补充。此外，LR White嵌入将每个mRNA的可用探针对识别位点的数量限制为一小部分。结果，RNA群体之间信号强度的差异反映了RNA三级结构的差异，并且我们表明该方法可用于探测mRNA的紧密性。我们应用LR White smFISH来回答一些与转拼，RNA颗粒和线粒体RNA编辑有关的悬而未决的问题，使用锥虫及其多功能RNA生物学作为模型系统。