Click-iT® Detection of RNA Synthesis
RNA like you’ve never seen it before!
Click-iT® RNA Imaging Snapshot
Click-iT® RNA assays are ideal for imaging global RNA synthesis in multiplexed analyses using either traditional fluorescence microscopy or high-content screening (HCS) techniques.
Additionally, you can capture newly synthesized RNA with the Click-iT® Nascent RNA Capture Kit.
Why Click-iT® Labeling and Detection is Perfect for RNA
| Click-iT® RNA Assays |
Click-iT® RNA assays employ an alkyne-modified nucleoside, EU (5-ethynyl uridine), that is fed to cells and incorporated into nascent RNA. The small size of the alkyne-tag (MW ~25) enables efficient incorporation by RNA polymerases without any apparent changes to the RNA levels of several housekeeping genes (Figure 1).
Detection utilizes an Alexa Fluor® azide, with a diminutive “footprint” (MW <1000) compared to an IgG antibody (MW ~150,000), that generates results in 30 minutes and completely eliminates long antibody incubations. Following EU incubation, simply fix, permeabilize, and perform the copper-catalyzed click reaction.
Figure 1. Housekeeping genes are unaffected in cells incubated with EU.
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Real-time RT-PCR was conducted to determine if EU incorporation affected housekeeping gene expression levels. RNA was isolated from NIH3T3 cells fed with 0.5 M EU and 1.0 mM EU using the TRIzol® LS reagent. Specific primer sets for hACtβ, hHprt1, and hPpib were used. The data clearly shows that mRNA levels of housekeeping genes do not change in the presence of the EU concentrations tested.
|Compatible with Cell Labeling Methods |
The Click-iT® RNA assays are completely compatible with other cell labeling methods such as immunocytochemistry and small-molecule labeling of cellular structures (Figure 2). The robustness and multiplexibility of this assay is ideal for toxicological profiling or interrogation of disease models using high-content imaging platforms.
Figure 2. Multiplex imaging with Click-iT® RNA assays.
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NIH3T3 cells were incubated with 1 mM EU, formaldehyde-fixed, and permeabilized with Triton® X-100. EU incorporated into newly synthesized RNA (red) in some cells was detectied using the Click-iT® RNA Alexa Fluor® 594 Imaging Kit. Tubulin (green) was detected with anti-tubulin mouse IgG9 and visualized with Alexa Fluor® 488 goat anti-mouse IgG. Nuclei (blue) were stained with Hoechst 33342.
Go Viral with Click-iT® RNA Assays
|Click-iT® RNA Imaging and HCS Kits |
The Click-iT® RNA imaging and HCS kits provide an exciting new alternative to the antibody-based BrU or BrUTP assays for studying viral RNA synthesis in infected cells (Figure 3). Little is understood about the sites of viral RNA synthesis, including the transport and fate of nascent RNA inside and outside the viral replication complex.1 Unlike BrUTP, EU does not require a transfection reagent and the small size of the Alexa Fluor® azide relative to the anti-BrdU antibody enables detection of both ssRNA and dsRNA.
The Click-iT® RNA imaging assays provide the tools necessary to produce quantitative and qualitative measurements of viral RNA synthesis, which will aid in understanding viral gene expression and genome replication, as well as its subcellular localization in host cells.
Figure 3. Subcellular localization of RNA in Vero cells.
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Vero cells were pretreated with 2 uM actinomycin D to inhibit host cell transcription, then infected with Tacaribe virus. Infected cells were incubated with 2 mM EU for 1 hr followed by cold methanol fixation and permeabilized with Triton® X-100. Nascent RNA (green) was detected with the Click-iT® RNA Alexa Fluor® 488 Imaging Kit. Viral nucleoprotein was detected with a directly labeled Alexa Fluor® 594 monoclonal antibody (red). Colocalization of EU and virus nucleoprotein indicated transcription sites in the host cells (yellow).
Find Your Click-iT® Detection Assay
- EdU, a new Thymidine Analogue for Labeling Proliferating Cells in the Nervous System—J. Neurosci Meth. 177, 122-130 (2009)
- Exploring RNA Transcription and turnover in-vivo by using Click Chemistry—PNAS 105, 15779-15784 (2008)
- Protein Synthesis in Distal Axons is not required for Growth Cone Responses to Guidance Cues—J. Neurosci. 29, 638-652 (2009)