RNA plays crucial role in coding, decoding and regulation of genes, and protein expression in all living cells. The ability to detect newly synthesized RNA or changes in RNA levels under various physiological conditions, or resulting from disease, environmental damage, or drug treatments is an important aspect of toxicological profiling. Many anti-cancer drugs inhibit transcription, and most transcription inhibitors have useful pharmacological properties. Assay Genie's Global RNA Synthesis Assay Kit provides a simple and robust tool for detection of global RNA transcription temporally and spatially or changes in RNA levels directly in living cells. De novo synthesized RNA can be detected with a simple procedure without the use of radiolabeling or antibodies. Our approach relies on the incorporation of cell permeable 5-EU (Ethynyl uridine) into nascent RNA, but not into DNA, instead of its natural uridine analog. 5-EU can be used as a replacement for BrU (5-Bromo-uridine) to measure de novo synthesized RNA in proliferating cells. Modified RNA is detected by click chemistry with azide-containing dye that enables for multiplex analyses with other probes, or detection of RNA-interactive proteins for deeper biological insights. Our kit provides sufficient materials for 100 assays for analysis by FACS or detection by fluorescence microscope. We include Actinomycin D, an inhibitor RNA synthesis that serves as an experimental control

Figure: Analysis of RNA biosynthesis in presence of Actinomycin D. (A) Jurkat cells (1X10 6 cells/ml) were pre-treated with vehicle (black line) or 1 X Actinomycin D (blue line) for 4 hours at 37°C prior to 1-hour incubation with RNA Label (red line). Cells were then processed for detection of de novo synthesized RNA according to the included protocol. Fluorescence measured in FL-2 channel clearly shows the inhibitory effect of Actinomycin D on RNA synthesis. (B) HeLa cells (10 5 cells/ ml) were pre-treated either with vehicle (top) or 1 Actinomycin D (bottom) for 4 hours at 37°C prior to 1-hour incubation with RNA Label. De novo synthesized RNA was detected by Fluorescence Microscope. Reduced red fluorescence in panel B confirms the inhibitory effect of Actinomycin D on RNA biosynthesis. Nuclear staining in both panels confirms that red fluorescence is the result of RNA Label incorporation.