FISH-Flow to quantify nascent and mature ribosomal RNA in mouse and human cells

Abstract

Summary: FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis.For complete details on the use and execution of this protocol, please refer to Antony et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Single Cell
• Flow Cytometry/Mass Cytometry
• Molecular Biology
• In Situ Hybridization