Using Tet-Off Cells and RNAi Knockdown to Assay mRNA Decay

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1720; p. 161
• Main Authors: Baird, Thomas D, Hogg, J Robert
• Format: Journal Article
• Language: English
• Published: United States 01.01.2018
• Subjects: Gene Expression Regulation; Gene Knockdown Techniques - methods; Genes, Reporter - genetics; Half-Life; HeLa Cells; Humans; Promoter Regions, Genetic - genetics; Recombinant Fusion Proteins - genetics; Repressor Proteins - genetics; RNA Interference; RNA Stability - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; Tetracycline - pharmacology; Trans-Activators - genetics; Transgenes - genetics; RNA stability; UPF1; RNA half-life; Nonsense-mediated mRNA decay (NMD); Tet-off cells; 3′ untranslated region; mRNA decay
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-7540-2_12

Cellular mRNA levels are determined by the competing forces of transcription and decay. A wide array of cellular mRNA decay pathways carry out RNA turnover either on a constitutive basis or in response to changing cellular conditions. Here, we outline a method to investigate mRNA decay that employs RNAi knockdown of known or putative decay factors in commercially available Tet-off cell systems. Reporter mRNAs of interest are expressed under the control of a tetracycline-regulated promoter, allowing pulse-chase mRNA decay assays to be conducted. Levels of reporter and constitutively expressed control RNAs throughout the decay assay time course are detected by traditional northern blot analysis and used to calculate mRNA half-lives. We describe the utility of this approach to study nonsense-mediated mRNA decay substrates and factors, but it can be readily adapted to investigate key mechanistic features that dictate the specificity and functions of any mRNA decay pathway.