Modified mRNA encoding Enhanced Green Fluorescent Protein (EGFP) with dual detection capabilities:

• Delivery tracking: Cy5 fluorophore (Ex/Em 650/670 nm) enables real-time visualization of mRNA uptake
• Expression monitoring: EGFP fluorescence (Ex/Em 488/507 nm) confirms protein translation

Incorporates N1-methyl-pseudouridine (m¹Ψ) for enhanced translational efficiency and reduced immunogenicity.

• 5' Cap: ARCA (m⁷G(5')ppp(5')G, Cap 0) or CleanCap AG (m⁷G(5')ppp(5')(2'-OMeA)pG, Cap 1)
• Nucleotide Modification: 1:3 ratio of Cy5-UTP to m¹Ψ-5'-triphosphate
• 3' Tail: 180 nt poly(A) sequence for mRNA stability
• Sequence Length: ~1,000 nt (including UTRs)

• Optimization of mRNA delivery systems
• Quantification of transfection efficiency
• Time-course studies of mRNA translation
• Control for mRNA-based experiments

• Concentration: 1.0 ± 0.2 µg/µL (UV quantification)
• Purity: >90% full-length by denaturing agarose gel electrophoresis
• Endotoxin: <0.1 EU/µg
• Recommended Storage: -80°C (aliquot to avoid freeze-thaw cycles)

For HEK293 cells:

1. Dilute 1 µg mRNA in 100 µL Opti-MEM®
2. Mix with 2 µL MessengerMAX™ reagent
3. Incubate 15 min at RT
4. Add to cells in 24-well plate

Full protocol: MessengerMAX™ Transfection Protocol

• Cap integrity analyzed by RNase H cleavage assay
• Poly(A) length verified by Northern blot
• Fluorescence intensity standardized against reference samples