Step-by-Step Guide for Research Applications
The rapid expansion of mRNA technology from vaccine development to gene editing, protein replacement, and cellular reprogramming has created an urgent need for small-scale, high-quality mRNA production workflows. Addressing this demand, a team led by Tim R. Mercer and Seth W. Cheetham from the University of Queensland has developed a standardized, equipment-agnostic protocol for mRNA preparation and lipid nanoparticle (LNP) delivery.
Published in Nature Protocols, their method provides a reproducible and practical solution for researchers conducting foundational experiments and preclinical validations.
Workflow Overview: From Sequence Design to LNP Delivery
The protocol outlines a step-by-step process, ensuring high-quality mRNA production and efficient LNP encapsulation:
Step 1: mRNA Primary Sequence Design
- mRNArchitect software is used to design mRNA constructs, incorporating:
5′ cap for stability and translation initiation
Optimized coding sequences (CDS) with codon optimization and uridine depletion
Untranslated regions (UTRs) for enhanced expression
PolyA tail for mRNA stability
- Secondary structure minimization is applied to improve translational efficiency.
Step 2: DNA Template Preparation
- PCR amplification generates high-purity DNA templates.
- Forward primers introduce a T7 promoter, while reverse primers encode the polyA tail, ensuring proper transcription initiation and termination.
- Compared to plasmid templates, PCR-derived templates eliminate endotoxin contamination and incomplete tailing, making them ideal for rapid iterations and small-scale production.
Step 3: DNA Template Quality Control
- UV spectrophotometry assesses yield and purity.
- Capillary gel electrophoresis (CGE) confirms specificity and integrity.
Step 4: mRNA Synthesis via In Vitro Transcription
- T7 RNA polymerase drives transcription.
- N1-methyl-pseudouridine (m1Ψ) replaces uridine to reduce immunogenicity and enhance stability.
- Co-transcriptional capping (Cap-1 structure) ensures proper 5′ modification.
- Magnesium ions, spermidine, and pyrophosphatase optimize reaction efficiency and prevent byproduct accumulation.
- Typical yields are achieved within 2–3 hours.
Step 5: mRNA Quality Control
- UV spectrophotometry measures concentration and purity.
- CGE evaluates mRNA integrity and absence of degradation.
Step 6: In Vitro Expression Validation
- HEK293T, HepG2, HuH7, and cortical neurons are transfected using MessengerMax.
- Dose-dependent expression is demonstrated using eGFP as a reporter.
Step 7: LNP Formulation
- Microfluidic mixing (e.g., NanoAssemblr Ignite) encapsulates mRNA in LNPs.
- Lipid composition includes:
SM-102 (ionizable lipid)
Cholesterol (structural stability)
DSPC (membrane integrity)
DMG-PEG2000 (stealth properties)
- Alternative manual mixing methods (e.g., T-mixer, herringbone mixer) ensure accessibility for labs without specialized equipment.
Step 8: LNP Quality Control
- Dynamic light scattering (DLS) measures particle size, PDI, and zeta potential.
- Ribogreen assay quantifies mRNA encapsulation efficiency (>90%).
- ELISA-based dsRNA detection ensures purity.
Key Advantages of the Protocol
- No viral elements or proprietary systems—fully compatible with standard molecular biology labs.
- Comprehensive QC standards, including:
mRNA concentration (≥4 mg/mL)
Encapsulation efficiency (>90%)
PDI (<0.2)
Zeta potential (±20 mV)
- Flexibility for research and preclinical applications, from functional studies to vaccine development.
Conclusion
This protocol provides researchers with a low-cost, high-quality, and rapid method for mRNA production and LNP formulation. By integrating best practices while avoiding expensive equipment dependencies, it offers a practical pathway for mRNA-based therapeutics, gene editing, and vaccine research.
Get Started: Whether you need custom mRNA design, in vitro transcription, or end-to-end LNP formulation services, our team provides solutions from sequence design to quality-controlled delivery.
Contact Us Today to discuss your research needs.
Interested in mRNA and LNP innovations? Explore more insights on our blog or contact us for custom mRNA solutions.
Professional Support for mRNA Vaccines
COVID-19 mRNA Vaccine Development Services
Nucleotide Modifications of mRNA
Nucleotide Modifications-Indispensable Elements for mRNA Vaccines
mRNA IVT byproduct – dsRNA Detection Service
For full details, refer to the original publication:
The design, manufacture and LNP formulation of mRNA for research use. Nature Protocols 2025. doi:10.1038/s41596-025-01174-4