RL450

Operating Instructions
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 Recommended concentration: 1 M.

2. Working Solution Preparation
2.1 Diluent: Nuclease-free water containing 5% DMSO (for in vitro RNA labeling); phosphate-buffered saline (PBS, 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: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedures
3.1 Sample Types^[1]:
3.1.1 In vitro RNA samples: Short single-stranded RNA (ssRNA) oligonucleotides, RNA molecular weight markers, and in vitro transcribed RNA.
3.1.2 Adherent cells (HeLa cells): RL420 and RL450 require cell fixation and permeabilization before labeling; RL480 (HY-D3364)/RL560 (HY-D3365) can be used for live cells without fixation.
3.2 Incubation Conditions:
3.2.1 In vitro RNA labeling: Incubate 50 mM RL450 with RNA at room temperature for 6 h (for high conversion rate) or 1 h (for rapid labeling).
3.2.2 Cell imaging: Incubate fixed/permeabilized HeLa cells with 20 μM RL450 for 30 min.
3.3 Washing Steps: No washing is required for live cell imaging with RL fluorescent dyes; for in vitro gel imaging, purify the labeled RNA by ethanol precipitation after incubation.

4. Control Setup
4.1 Set up the following negative controls:
4.1.1 DNA oligonucleotides with identical sequences to the target RNA, to verify RNA selectivity.
4.1.2 The unactivated carboxylic acid precursor of RL450 (CA450), to verify that covalent labeling is essential for fluorescence generation.
4.1.3 Template-free control for real-time RNA synthesis monitoring, to eliminate non-specific fluorescence.
4.2 Positive controls are used to verify the validity of the experimental system; blank controls are used to exclude fluorescence interference from reagents.

5. Detection and Analysis
5.1 Instruments: Fluorescence spectrophotometer (for in vitro solution analysis); laser confocal fluorescence microscope (for cell imaging); real-time PCR instrument (for real-time RNA synthesis monitoring); gel fluorescence imager (for PAGE gel imaging).
5.1.1 Excitation/emission wavelengths: λex = 450 nm, λem = 497 nm (free RL450); λex = 447 nm, λem = 497 nm (RL450-labeled RNA).
5.2 Result Analysis:
5.2.1 Changes in fluorescence intensity: After covalent labeling with RNA, the fluorescence intensity can increase by up to 970-fold, while the fluorescence is extremely weak in the presence of DNA, small molecules or unreacted dyes.
5.2.2 Fluorescence localization: In fixed cells, the fluorescence signal from RL450 labeling is significantly enriched in RNA-rich nucleolar regions.
5.2.3 Color change: RL450-labeled RNA shows cyan fluorescence.

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