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原初微小RNA在分离细胞核中的荧光原位定位

Fluorescence in situ Localization of Pri-miRNAs in Isolated Nuclei.

作者信息

Gulanicz Tomasz, Zienkiewicz Agnieszka, Zienkiewicz Krzysztof, Kasprowicz-Maluski Anna, Szweykowska-Kulinska Zofia, Jarmolowski Artur

机构信息

Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Torun, Poland.

Department of Molecular and Cellular Biology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland.

出版信息

Bio Protoc. 2023 Sep 20;13(18):e4824. doi: 10.21769/BioProtoc.4824.

DOI:10.21769/BioProtoc.4824
PMID:37753471
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10518776/
Abstract

Here, we present an approach combining fluorescence in situ hybridization (FISH) and immunolabeling for localization of pri-miRNAs in isolated nuclei of A. thaliana. The presented method utilizes specific DNA oligonucleotide probes, modified by addition of digoxigenin-labeled deoxynucleotides to its 3' hydroxyl terminus by terminal deoxynucleotidyl transferase (TdT). The probes are then detected by immunolabeling of digoxigenin (DIG) using specific fluorescent-labeled antibodies to visualize hybridized probes. Recently, we have applied this method to localize pri-miRNA156a, pri-miRNA163, pri-miRNA393a, and pri-miRNA414 in the nuclei isolated from leaves of 4-week-old A. thaliana. The present approach can be easily implemented to analyze nuclear distribution of diverse RNA classes, including mRNAs and pri-miRNAs in isolated fixed cells or nuclei from plant.

摘要

在此，我们展示了一种将荧光原位杂交（FISH）与免疫标记相结合的方法，用于在拟南芥分离细胞核中定位初级微小RNA（pri-miRNA）。所展示的方法利用特定的DNA寡核苷酸探针，通过末端脱氧核苷酸转移酶（TdT）在其3'羟基末端添加地高辛标记的脱氧核苷酸进行修饰。然后使用针对地高辛（DIG）的特异性荧光标记抗体通过免疫标记来检测探针，以可视化杂交探针。最近，我们已应用此方法在从4周龄拟南芥叶片分离的细胞核中定位pri-miRNA156a、pri-miRNA163、pri-miRNA393a和pri-miRNA414。本方法可轻松用于分析包括植物分离的固定细胞或细胞核中的信使核糖核酸（mRNA）和pri-miRNA等多种RNA类别的核分布。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ad06/10518776/c397abe68f78/BioProtoc-13-18-4824-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ad06/10518776/288628c57dc4/BioProtoc-13-18-4824-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ad06/10518776/dbc35f035558/BioProtoc-13-18-4824-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ad06/10518776/c397abe68f78/BioProtoc-13-18-4824-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ad06/10518776/288628c57dc4/BioProtoc-13-18-4824-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ad06/10518776/dbc35f035558/BioProtoc-13-18-4824-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ad06/10518776/c397abe68f78/BioProtoc-13-18-4824-g003.jpg

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