Bio-Protocol (Sep 2023)

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

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

DOI
https://doi.org/10.21769/BioProtoc.4824
Journal volume & issue
Vol. 13, no. 18

Abstract

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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.