Fluorescence in situ Localization of Pri-miRNAs in Isolated
Confocal microscopy
Digoxigenin
Fluorescent in situ hybridization
Immunolabeling
Pri-miRNA
Journal
Bio-protocol
ISSN: 2331-8325
Titre abrégé: Bio Protoc
Pays: United States
ID NLM: 101635102
Informations de publication
Date de publication:
20 Sep 2023
20 Sep 2023
Historique:
received:
20
03
2023
revised:
25
06
2023
accepted:
17
07
2023
medline:
27
9
2023
pubmed:
27
9
2023
entrez:
27
9
2023
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 37753471
doi: 10.21769/BioProtoc.4824
pii: e4824
pmc: PMC10518776
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e4824Informations de copyright
©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.
Déclaration de conflit d'intérêts
Competing interestsThere are no conflicts of interest or competing interests.
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