The Click-iT® RNA Alexa Fluor® 488 Imaging Kit enables detection of global RNA transcription temporally and spatially in cells and tissues (PNAS (2008) 105:15779-84). The ability to detect newly synthesized RNA or changes in RNA levels resulting from disease, environmental damage or drug treatments is an important aspect of toxicological profiling. Utilizing an alkyne-modified nucleoside, 5-ethynyl uridine (EU), and powerful click chemistry, newly synthesized RNA can be detected without the use of radioactivity or antibodies with a simple, two-step procedure. In step one, the alkyne-containing nucleoside is fed to cells or animals and actively incorporated into nascent RNA. The small size of the tag enables efficient incorporation of the modified nucleoside into RNA, but not into DNA. Detection utilizes the chemoselective ligation or “click" reaction between and azide and an alkyne where the modified RNA is detected with a corresponding azide-containing dye. With its dimunitive “footprint", the Click-iT® detection molecule can easily penetrate complex samples and leaves open the possibility of multiplex analyses with other probes, including antibodies for the detection of RNA-interactive proteins for deeper biological insights.
For Research Use Only. Not for use in diagnostic procedures.
New RNA Synthesis Assay
Alexa Fluor Dyes
Alexa Fluor™, Click-iT™, Molecular Probes™
For Use With (Equipment)
Confocal Microscope, Fluorescence Microscope
Label or Dye
Alexa Fluor™ 488
Contents & Storage
The kit contains sufficient material for 25, 18 X 18 coverslips. Store at 2-6°C, dessicate and protect from light.
Multiplex imaging of NIH3T3 cells.
Housekeeping genes are unaffected in cells incubated with EU. RNA was isolated using TRIzol® LS reagent (Cat. No. 102906-010) from NIH3T3 cells fed with EU. RNA concentrations were determined for each sample, and then 250 ng of RNA and specific primer sets for hACtß, hHprt1, and hPpib were used in a reaction volume of 50 µL to perform one-step qPCR using the SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit (Cat. No.11736-051) and the Mx3000P system (Stratagene).
Subcellular localization of RNA in Vero cells. Vero cells were pretreated with actinomycin D, then infected with Tacaribe virus. Infected cells were incubated with EU, fixed and then permeabilized. Nascent RNA (green) was detected with the Click-iT® RNA Alexa Fluor® 488 Imaging Kit (Cat. No. C10329). Viral nucleoprotein was detected with an Alexa Fluor® 594 antibody (red). Colocalization of EU and virus nucleoprotein indicates transcription sites in the host cells (yellow).