Fluorescence In Situ Hybridization of Small Non-Coding RNAs.
Fiche publication
Date publication
janvier 2021
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr BEHM-ANSMANT Isabelle
Tous les auteurs :
Vautrot V, Heckler G, Aigueperse C, Behm-Ansmant I
Lien Pubmed
Résumé
The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor 488 conjugate. A protocol allowing efficient coupling of RNA-FISH and protein detection by immunofluorescence is also described as well as the bioinformatics pipeline, Substructure Analyzer, we recently developed to automate fluorescence signal analysis.
Mots clés
Fluorescence, LNA probe, LNA-FISH, RNA intracellular localization, RNA-FISH, Substructure analyzer
Référence
Methods Mol Biol. 2021 ;2300:73-85