Lee ChangHwan, Seidel Hannah S, Lynch Tina R, Sorensen Erika B, Crittenden Sarah L, Kimble Judith
Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Bio Protoc. 2017 Jun 20;7(12):e2357. doi: 10.21769/BioProtoc.2357.
Single-molecule RNA fluorescence hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj , 2008 ; Lee , 2016a ). We adapted this technique to visualize RNAs in the whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee , 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for extruded gonads.
单分子RNA荧光杂交（smFISH）是一种利用针对目标RNA的多个荧光标记寡核苷酸探针来可视化单个RNA分子的技术（Raj，2008年；Lee，2016年a）。我们采用了这项技术来观察成年线虫整体或其生殖系中的RNA，这使得能够同时记录活跃转录位点处的新生转录本和细胞质中的成熟mRNA（Lee，2013年和2016年b）。在此，我们描述了针对挤出性腺优化的smFISH程序、试剂和显微镜设置的每个步骤。
