Overview

  • Product name

    RNA Synthesis Assay Kit
  • Detection method

    Fluorescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based
  • Species reactivity

    Reacts with: Other species, Mammals
  • Product overview

    RNA Synthesis Assay Kit (ab228561) 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. The 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. The 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.

  • Notes

    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.

  • Platform

    Flow cytometer, Fluorescence microscope

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    Actinomycin D (100X) 1 x 10µl
    Copper Reagent (100X) 1 x 100µl
    DAPI (1000X) 1 x 20µl
    Fixative Solution 1 x 10ml
    Fluorescent Azide (100X) 1 x 100µl
    Permeabilization Buffer (10X) 1 x 25ml
    Reducing Agent (20X) Yellow 1 x 500µl
    RNA Label (100X) 1 x 100µl
    Wash Buffer (10X) 1 x 25ml

Images

  • Jurkat cells (1X106 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.

  • HeLa cells (105 cells/ mL) were pre-treated either with vehicle (top) or 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.

Protocols

References

ab228561 has not yet been referenced specifically in any publications.

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