Click-iT® Nascent RNA Capture benefits:
- Capture and characterize newly synthesized RNA transcripts
- Investigate RNA turnover
- Simple procedure, no antibodies, no radioactivity
- Compatible with high-resolution gene expression applications
What is Click-iT® Nascent RNA Capture?
Click-iT® technology is a nonradioactive method for live cell metabolic labeling. This method is based on the bioorthogonal click chemistry reaction, which enables the metabolic incorporation of ethynyl uridine (EU) – a "clickable" ribonucleoside – onto RNA during nascent RNA synthesis. Biotin is then “clicked” onto the nascent chain and strepavidin magnetic beads used to capture the transcriptome of that sample. 40 reactions in one kit.
Enabling High-Resolution Analysis of the Transcriptome
Analysis of newly transcribed RNA is of critical importance in investigating the transcriptome. The unique Click-iT® Nascent RNA Capture Kit is the first commercial method that allows you to isolate and analyze new RNA transcripts from metabolically active cell models.
How it Works
Magnetic Beads efficiently Capture with EU-Nascent RNA
The EU-nascent RNA is efficiently captured on magnetic beads for subsequent cDNA synthesis and qPCR, microarray analysis, and/or sequencing (Figure 1). Our data demonstrates metabolic labeling with EU has no effect on cell health or viability over a time course of 4 hours (Figure 2 and 3).
The ability to metabolically label newly transcribed RNA not only lends valuable information on the dynamics of gene expression, but also enables the researcher to perform pulse/chase experiments to determine RNA transcript stability without the use of radioactive compounds (Figure 4).
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Figure 1—Schematic of Click-iT® Nascent RNA Capture Kit procedure.
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Figure 2 — EU does not cause apoptosis or unnatural cell death. Jurkat cells were fed 200 μM EU for 4 hours and the cell toxicity was determined using Alexa Fluor® 488 Annexin V/propidium iodide stain and analyzed by flow cytometry. The results showed no differences in live, apoptotic or dead cell populations between EU treated and control.
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Figure 3 — Effect of EU incorporation on the global transcriptome analyzed by microarray analysis. Microarray analysis of total RNA from EU treated and non-treated A549 cells. Microarray analysis was carried out using the Ambion® WTEK kit and the Affymetrix fragmentation and labeling kit on an Affymetrix GeneChip® Human Exon 1.0 ST array. Partek® Genomics Suite™ software was used to process the data using standard RMA normalization and perform statistical tests. A tight correlation indicates no difference in gene expression between EU treated and non-treated cells. |
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Figure 4 — Kinetics of RNA decay determined using the Click-iT® Nascent RNA Capture Kit. Jurkat cells were pulsed with EU for 24 hours followed by replacement of medium without EU (chase) or dilution of cells in a medium without EU. Total RNA was isolated and subjected to nascent RNA capture and analyzed by real-time PCR (A) for the genes indicated showing a loss of labeled RNA after the 24 hour chase (B).
Find Your Click-iT® Detection Assay
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