Cellular RNA levels are orchestrated by highly regulated processes involving RNA synthesis (transcription), processing (e.g., splicing, polyadenylation, transport), and degradation. Profiling these changes provides valuable information on the regulation of gene expression. Total cellular RNA is a poor template for revealing short-term changes in gene expression, alterations in RNA decay rates, and the kinetics of RNA processing as well as the differentiation thereof. Here, University of Cambridge researchers describe the metabolic labeling and purification of newly transcribed RNA with 4-thiouridine, by which these limitations are overcome.
Schematic overview on 4sU-tagging
Metabolic labeling of newly transcribed is initiated when 4sU is added to the cell culture medium . Following isolation of total cellular RNA, 4sU residues in newly transcribed RNA are thiol-specifically biotinylated. This allows separation of total RNA into newly transcribed (4sU-RNA) and unlabeled pre-existing RNA using streptavidin -coated magnetic beads. 4sU-RNA is recovered from the beads by adding a reducing agent, which cleaves the disulfide bond between 4sU and biotin . 4sU-RNA is recovered by column purification or isopropanol/ethanol precipitation