Fluorescence In Situ Imaging of Dendritic RNAs at Single-Molecule Resolution.
Fluorescence In Situ Imaging of Dendritic RNAs at Single-Molecule Resolution.
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DOI：
10.1002/cpns.79
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发表时间：
2019-09-01
影响因子：
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通讯作者：
Tyagi, Sanjay
中科院分区：
文献类型：
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作者：
Batish, Mona;Tyagi, Sanjay
RNA定位是基因调控的重要步骤。固定和活细胞中的RNA成像提供了有关细胞中RNA分布的背景信息。在这里，我们提供了详细的协议进行单分子荧光原位杂交（smFISH）。smFISH在固定的神经元细胞中以单分子分辨率检测mRNA分子，每个mRNA使用50个小寡核苷酸探针。该技术已成功地应用于了解RNA在各种生物系统中的定位和分布，从果蝇到哺乳动物神经系统。探针足够小，可以结合到结构化RNA或RNA-蛋白质复合物的一部分，从而占总RNA的85%;这使得灵敏度水平与定量实时PCR相当，但具有解剖分辨率。John Wiley & Sons，Inc._关于我们
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.