Detection and Quantification of Multiple RNA Sequences Using Emerging Ultrasensitive Fluorescent In Situ Hybridization Techniques.
Detection and Quantification of Multiple RNA Sequences Using Emerging Ultrasensitive Fluorescent In Situ Hybridization Techniques.
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DOI：
10.1002/cpns.63
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发表时间：
2019-04-01
影响因子：
--
通讯作者：
Buonanno, Andres
中科院分区：
文献类型：
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作者：
Erben, Larissa;Buonanno, Andres
使用RNAscope荧光检测转录本已迅速成为神经科学中的标准原位杂交（ISH）方法，自2012年推出以来已有400多篇出版物。RNAscope的灵敏度和特异性允许同时检测单细胞中多达三种低丰度mRNA（即，多路复用），并且与其他ISH技术相反，RNAscope在1天内进行。BaseScope是一种较新的超灵敏平台，使用改进的单寡核苷酸探针对（50个碱基）扩增化学。该技术允许通过短外显子区分单核苷酸多态性或剪接变体。BaseScope目前的局限性是表达分析限于单个基因（即，单路复用）。本文概述了RNAscope和BaseScope在神经元组织中的详细方案。我们讨论了如何使用新鲜冷冻或福尔马林固定石蜡包埋切片，以及分离培养的神经元进行ISH实验。我们还概述了如何从杂交组织切片获得定量数据。John Wiley & Sons，Inc._关于我们
Fluorescent detection of transcripts using RNAscope has quickly become a standard in situ hybridization (ISH) approach in neuroscience with over 400 publications since its introduction in 2012. RNAscope's sensitivity and specificity allow the simultaneously detection of up to three low abundance mRNAs in single cells (i.e., multiplexing) and, in contrast to other ISH techniques, RNAscope is performed in 1 day. BaseScope, a newer ultrasensitive platform, uses improved amplification chemistry of single oligonucleotide probe pairs (50 bases). This technique allows discrimination of single nucleotide polymorphisms or splice variants that differ by short exons. A present limitation of BaseScope is that expression analysis is limited to a single gene (i.e., single-plexing). This article outlines detailed protocols for both RNAscope and BaseScope in neuronal tissue. We discuss how to perform ISH experiments using either fresh-frozen or formalin-fixed paraffin-embedded sections, as well as dissociated cultured neurons. We also outline how to obtain quantitative data from hybridized tissue sections. © 2019 by John Wiley & Sons, Inc.