Emission (nm): 545
Excitation (nm): 490The major challenge to analyze RNA in live cells is the interferences caused by DNA. To address these difficulties, AAT Bioquest has developed the StrandBrite™ RNA Green, an excellent RNA-selective probe that generates significantly enhanced green fluorescence upon binding to RNA. It has been successfully used for flow cytometric analysis of live cells. StrandBrite™ RNA Green readily gets into live cells. It has the excitation/emission of 490/540 nm. In the DNase digest test, no significant change of fluorescence intensity in fixed cells stained with StrandBrite RNA Green was observed. In contrast, after RNase digestion, the initial fluorescence signal decreased immediately. These results indicate that initial fluorescence signal was generated from the specific interaction of StrandBrite RNA Green with RNA in cells. Short exposure of live cells to antinomycin D did cause inhibition of RNA synthesis during 6 hours after drug removal in a dose-dependent manner. These data demonstrate that StrandBrite RNA Green is a sensitive RNA-selective dye for staining nucleolar RNA in live and fixed cells. StrandBrite RNA Green has less DNA interferences than the commonly used SYTO® RNASelect™ dye. StrandBrite™ RNA Green is a highly RNA-selective fluorescent probe. Due to its excellent cell permeability and spectral properties, it has been successfully used for flow cytometric RNA analysis and fluorescence microscope in live cells. It can be well excited with the 488 nm blue laser and monitored in FITC channel. StrandBrite™ RNA Green provides a valuable method for identifying and labeling cells with a single incubation step and can discriminate RNA from DNA with better selectivity than the commonly used SYTO® RNASelect™.
Fluorescence Microplate Reader
Excitation (nm): 490
Emission (nm): 540
Cutoff (nm): 515
Recommended Plate: Solid Black
Component A: StrandBrite™ RNA Green: 1 vial (50µL, 200X in DMSO)
Component B: 10X Assay Buffer: 1 bottle (5 mL)
Component C: Ribosomal RNA Standard: 1 vial (20µL, 2mg/mL)
At a glance
- Prepare StrandBrite™ RNA Green working solution (100 µL)
- Add RNA standards or test samples (100 µL)
- Incubate at room temperature for 2 - 5 minutes
- Monitor the fluorescence intensity at Ex/Em = 490/540 nm (Cutoff = 515 nm)
The following protocol is an example for quantifying RNA with StrandBrite™ Green Fluorimetric RNA Quantitation Kit. Allow all the components to warm to room temperature before opening. To prevent RNase contamination of the StrandBrite™ reagent and kit components, always use clean disposable gloves while handling all materials. Use nucleasefree water, and sterile, disposable polypropylene plastic ware for reagent preparation. No data are available addressing the mutagenicity or toxicity of StrandBrite™ RNA Green. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution.
Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1. Assay buffer (1X):
Dilute 10X Assay Buffer (Component B) in sterile, distilled, nuclease-free water to make 1X Assay Buffer.
Preparation of standard solution
Add 10 µL of 2 mg/mL Ribosomal RNA Standard (Component C) to 990 µL of 1X Assay buffer to make 20 µg/mL RNA standard solution (RS7). Take 20 µg/mL RNA standard solution (RS7) and perform 1:2 serial dilutions in 1X Assay buffer to get serially diluted RNA standards (RS6 - RS1).
Preparation of working solution
Add 10 μL of StrandBrite™ RNA Green (Component A) into 1.99 mL of 1X Assay buffer to make StrandBrite™ RNA Green working solution. Protect StrandBrite™ RNA Green working solution from light by covering it with foil or placing it in the dark. Note: We recommend preparing this solution in a plastic container rather than glass, as the dye may adsorb to glass surfaces. For best results, this solution should be used within a few hours of its preparation.
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