Zirkel Anne, Papantonis Argyris
Chromatin Systems Biology Laboratory, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
Methods Mol Biol. 2018;1724:69-75. doi: 10.1007/978-1-4939-7562-4_6.
Fluorescence in situ hybridization (FISH) coupled to high-resolution microscopy is a powerful method for analyzing the subcellular localization of RNA. However, the detection of circular RNAs (circRNAs) using microscopy is challenging because the only feature of a circRNA that can be used for the probe design is its junction. Circular RNAs are expressed at varying levels, and for their efficient monitoring by FISH, background fluorescence levels need to be kept low. Here, we describe a FISH protocol coupled to high-precision localizations using a single fluorescently labeled probe spanning the circRNA junction; this allows circRNA detection in mammalian cells with high signal-to-noise ratios.
荧光原位杂交（FISH）与高分辨率显微镜相结合是分析RNA亚细胞定位的有力方法。然而，使用显微镜检测环状RNA（circRNA）具有挑战性，因为可用于探针设计的circRNA唯一特征是其连接点。环状RNA的表达水平各不相同，为了通过FISH对其进行有效监测，背景荧光水平需要保持较低。在这里，我们描述了一种FISH方案，该方案使用跨越circRNA连接点的单个荧光标记探针与高精度定位相结合；这使得能够在哺乳动物细胞中以高信噪比检测circRNA。
