RL480

Operating Instructions
(The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs)

1. Stock Solution Preparation
1.1 Solvent: Anhydrous dimethyl sulfoxide (DMSO).
1.2 Concentration recommendation: 1 M.

2. Working Solution Preparation
2.1 Diluent: Nuclease-free water containing 5% DMSO for in vitro reactions; phosphate-buffered saline (PBS, pH 7.4) for biological selectivity assays; cell culture medium for cell imaging.
2.2 Working concentration: 20 μM for cell imaging.
2.3 Notes: The working solution concentration can be adjusted as needed; prepare fresh and use immediately.

3. Staining Procedures
3.1 Sample types:
3.1.1 In vitro RNA samples: Single-stranded RNA (ssRNA) oligonucleotides, in vitro transcribed RNA, RNA molecular weight markers.
3.1.2 Cell samples: Adherent HeLa cells.
3.2 Incubation conditions:
3.2.1 In vitro RNA labeling: Incubate with 50 mM RL480 at room temperature for 24 h to achieve maximum adduct formation; 1 h of incubation is sufficient for gel imaging.
3.2.2 Cell imaging: Incubate live HeLa cells with 20 μM RL480 for 30 min.
3.3 Washing steps: No washing is required for live cell imaging; in vitro labeled RNA needs to be purified by ethanol precipitation before gel analysis.

4. Control Settings
4.1 Set a no-template control for real-time RNA synthesis monitoring.
4.2 Set a non-reactive carboxylic acid precursor (CA480) control for cell imaging.
4.3 Set a control using ssDNA with an identical sequence to verify RNA selectivity.
4.4 Set an RNase digestion control to confirm RNA-specific fluorescence.

5. Detection and Analysis
5.1 Instruments:
5.1.1 Fluorescence spectrophotometer: Ex = 480 nm, Em = 514 nm.
5.1.2 Real-time PCR instrument: Ex = 470 nm, Em = 520 nm.
5.1.3 Fluorescence gel imaging system: Ex = 480 nm, Em = 514 nm.
5.1.4 Laser confocal fluorescence microscope: Ex = 480 nm, Em = 514 nm.
5.2 Result analysis:
5.2.1 Fluorescence intensity change: The fluorescence intensity can increase by up to 320-fold after covalent binding to RNA, while the fluorescence is extremely weak in the presence of DNA, proteins or small-molecule nucleophiles.
5.2.2 Fluorescence localization: Signals are enriched in the nucleoli and cytoplasm of HeLa cells; colocalize with the stress granule marker G3BP1 in stressed cells.
5.2.3 Color change: Green fluorescence is exhibited after RNA labeling.