植物小RNA荧光原位杂交实验方法

doi:10.11983/CBB21057

摘要/Abstract

摘要： 小RNA是对植物生长发育十分重要的一类小分子核苷酸, 在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能, 而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小 RNA进行定性或半定量分析的技术, 目前该技术已经在动物体内被广泛应用, 而在植物体内的应用还比较少。该文详细介绍了基于超高分辨率显微镜的sRNA-FISH的具体操作流程以及注意事项, 该技术可用于探究植物组织内小RNA的表达与定位。

关键词: 荧光原位杂交, 小RNA, 植物

Abstract: Small RNAs are a type of small nucleotide molecules that are essential for plant growth and development, playing a key role in variety of life processes and in response to stresses. Research on the location of small RNAs could discover their functions in plants. Small RNA FISH is a qualitative or semi-quantitative analysis of small RNA in organisms by fluorescence detection technology. At present, this technology has been widely used in animals, but it is still less applied in plants. This article introduces the specific operation procedures and attentions based on ultra-high resolution microscopy that combines locked nucleic acid (LNA) probe in situ hybridization with immunofluorescence. This protocol can be used to detect the expression and localization of small RNA in plant tissues.

Key words: fluorescent in situ hybridization, small RNA, plants

胡滨滨, 薛治慧, 张翠. 植物小RNA荧光原位杂交实验方法. 植物学报, 2021, 56(3): 330-338.

Binbin Hu, Zhihui Xue, Cui Zhang. Protocols for Small RNA FISH in Plants. Chinese Bulletin of Botany, 2021, 56(3): 330-338.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21057

https://www.chinbullbotany.com/CN/Y2021/V56/I3/330

图/表 2

图1 小RNA荧光原位杂交实验流程图 (A) 探针设计; (B) 石蜡切片; (C) 探针杂交; (D) 一抗孵育; (E) 二抗孵育; (F) 光谱拆分以及激光共聚焦显微成像

Figure 1 Outlines of small RNA fluorescent in situ hybridization (A) Probe design; (B) Paraffin sectioning; (C) Probe hybridization; (D) Primary antibody incubation; (E) Secondary antibody incubation; (F) Spectral unmixing and imaging by confocal microscope

图2 小RNA FISH效果示例 (A) miR165/166反义寡核苷酸探针; (B) 阴性对照: miR165/ 166顺义寡核苷酸探针。Bars=50 μm

Figure 2 Schematic diagram of sRNA FISH (A) Antisense probe of miR165/166; (B) Negative control: Sense probe of miR165/166. Bars=50 μm

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