Fluorescence in situ Localization of Pri-miRNAs in Isolated Arabidopsis thaliana Nuclei

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...

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Bibliographic Details
Published in:Bio-protocol 2023-09, Vol.13 (18), p.e4824-e4824
Main Authors: Gulanicz, Tomasz, Zienkiewicz, Agnieszka, Zienkiewicz, Krzysztof, Kasprowicz-Maluski, Anna, Szweykowska-Kulinska, Zofia, Jarmolowski, Artur
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Language:English
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Biology
Methods
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Summary: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.
ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.4824