RL420

Operating Instructions
(The following is a recommended experimental protocol for guidance only; adjustments are required based on 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 RNA labeling); phosphate-buffered saline (1×, pH = 7.4) (for cell imaging).
2.2 Working concentration: 50 mM (for in vitro RNA labeling); 20 μM (for cell imaging).
2.3 Notes: The working solution concentration can be adjusted as needed; prepare fresh immediately before use.

3. Staining Procedures
3.1 Sample types^[1]:
3.1.1 In vitro samples: Single-stranded RNA (ssRNA) oligonucleotides, RNA molecular weight markers, in vitro transcription reaction mixtures.
3.1.2 Cell samples:
3.1.2.1 Adherent cells (HeLa cells): Fixation and permeabilization are required for labeling with RL420; live cell labeling with RL480 (HY-D3364) / RL560 (HY-D3365) does not require fixation/permeabilization.
3.2 Incubation conditions:
3.2.1 In vitro RNA labeling: RL420 requires incubation at room temperature for 6 h.
3.2.2 Cell labeling: Incubate live cells with 20 μM RL480/RL560 for 30 min; incubate fixed/permeabilized cells with RL420 for 30 min.
3.3 Washing steps: No washing is required for cell imaging; RNA labeled in vitro needs to be purified by ethanol precipitation before gel analysis.

4. Control Setup
4.1 Set up an ssDNA control with an identical sequence to the RNA to verify RNA selectivity.
4.2 Set up a non-reactive carboxylic acid precursor (CA420) control to verify that fluorescence depends on covalent labeling.
4.3 Set up a no-template control for real-time monitoring of RNA synthesis processes.
4.4 A positive control is used to verify the validity of the experimental system; a blank control is used to exclude fluorescence interference from the reagents themselves.

5. Detection and Analysis
5.1 Instruments:
5.1.1 Fluorescence spectrophotometer: Excitation (Ex) = 420 nm, emission (Em) = 452 nm (for RNA labeled with RL420).
5.1.2 MALDI-TOF mass spectrometer: Used to confirm the formation of covalent adducts.
5.1.3 Fluorescence gel imaging system: Used for PAGE gel imaging of labeled RNA.
5.1.4 Laser confocal fluorescence microscope: Excitation (Ex) = 420 nm, collect blue channel signals (for fixed cells labeled with RL420).
5.2 Result analysis:
5.2.1 Fluorescence intensity change: The fluorescence intensity of RNA labeled with RL420 increases up to 150-fold; only extremely weak fluorescence is produced when interacting with DNA or small molecule interferents.
5.2.2 Fluorescence localization: In fixed cells labeled with RL420, fluorescence signals accumulate in the nucleolus (an RNA-rich region).
5.2.3 Color change: RNA labeled with RL420 emits blue fluorescence.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.