Haimovich Gal, Gerst Jeffrey E
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Methods Mol Biol. 2019;2038:109-129. doi: 10.1007/978-1-4939-9674-2_8.
In eukaryotic cells, a small percentage of mRNA molecules can undergo transfer from one cell to another. mRNA transfer occurs primarily via membrane nanotubes, which are long thin protrusions that are produced by numerous cell types and can connect cells that can be up to hundreds of microns apart. Potentially, mRNAs might also transfer via extracellular vesicles (EVs). Here we describe a method to detect transferred mRNA in cocultures of two different cell types and to distinguish between nanotube- and EVs-mediated transfer. This method uses single molecule fluorescent in situ hybridization (smFISH) to provide an accurate and quantitative detection of transferred mRNA molecules and their subcellular localization. Following the guidelines presented here will allow the user to investigate mRNA transfer of most transcripts in any co-culture system. In addition, we present modifications that improve nanotube preservation during the smFISH procedure.
在真核细胞中，一小部分mRNA分子能够从一个细胞转移到另一个细胞。mRNA转移主要通过膜纳米管进行，膜纳米管是由多种细胞类型产生的细长突出物，能够连接相距可达数百微米的细胞。mRNA也有可能通过细胞外囊泡（EVs）进行转移。在此，我们描述了一种方法，用于检测两种不同细胞类型共培养物中转移的mRNA，并区分纳米管介导和EVs介导的转移。该方法使用单分子荧光原位杂交（smFISH）来准确、定量地检测转移的mRNA分子及其亚细胞定位。遵循此处给出的指南将使使用者能够在任何共培养系统中研究大多数转录本的mRNA转移。此外，我们还介绍了一些改进措施，可在smFISH过程中更好地保存纳米管。
