RNA Detection

Anupam A. Sawant, Sanjeev Galande, Seergazhi G. Srivatsan

Robust RNA labeling and imaging methods that enable the understanding of cellular RNA biogenesis and function are highly desired. In this context, we describe a practical chemical labeling method based on a bioorthogonal reaction, namely, azide-alkyne cycloaddition reaction, which facilitates the fluorescence imaging of newly transcribed RNA in both fixed and live cells. This strategy involves the transfection of an azide-modified UTP analog (AMUTP) into mammalian cells, which gets specifically incorporated into RNA transcripts by RNA polymerases present inside the cells. Subsequent posttranscriptional click reaction between azide-labeled RNA transcripts and a fluorescent alkyne substrate enables the imaging of newly synthesized RNA in cells by confocal microscopy. Typically, 50 μM to 1 mM of AMUTP and a transfection time of 15-60 min produce significant fluorescence signal from labeled RNA transcripts in cells.