Fluorescence in situ hybridization of small non-coding RNAs

Abstract

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 in order to study the expression and localization of satellite III RNAs. This specific class of non-coding RNAs is expressed in response to various cellular stresses including heat shock. This 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 in this chapter.