Batish Mona, Tyagi Sanjay
Department of Medical and Molecular Sciences and Department of Biological Sciences, University of Delaware, Newark, Delaware.
Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, New Jersey.
Curr Protoc Neurosci. 2019 Sep;89(1):e79. doi: 10.1002/cpns.79.
RNA localization is an important step in gene regulation. Imaging RNAs in fixed and live cells provides contextual information about RNA distribution in the cells. Here, we provide detailed protocols for performing single-molecule fluorescence in situ hybridization (smFISH). smFISH detects mRNA molecules at single-molecule resolution in fixed neuronal cells using ∼50 small oligonucleotide probes for each mRNA. The technique has been successfully applied to understand RNA localization and distribution in various biological systems, ranging from Drosophila to the mammalian nervous system. The probes are small enough to bind to structured RNAs or RNAs that are part of RNA-protein complexes, thereby accounting for ∼85% of the total RNA; this enables a level of sensitivity equivalent to that of quantitative real-time PCR, but with anatomical resolution. © 2019 by John Wiley & Sons, Inc.
RNA定位是基因调控中的重要一步。对固定细胞和活细胞中的RNA进行成像可提供有关RNA在细胞中分布的背景信息。在此，我们提供了用于进行单分子荧光原位杂交（smFISH）的详细方案。smFISH使用针对每个mRNA的约50个小寡核苷酸探针，以单分子分辨率检测固定神经元细胞中的mRNA分子。该技术已成功应用于理解从果蝇到哺乳动物神经系统等各种生物系统中的RNA定位和分布。这些探针足够小，可以与结构化RNA或作为RNA-蛋白质复合物一部分的RNA结合，从而占总RNA的约85%；这使得灵敏度水平与定量实时PCR相当，但具有解剖学分辨率。© 2019约翰威立国际出版公司。
