The mRNA Development Workflow:
From Plasmid to IVT, Capping, Purification, LNP and QC
mRNA development involves much more than a T7 RNA polymerase reaction. From DNA template preparation to transcription, capping, purification, delivery, and quality control, each stage requires the right raw materials and analytical tools.
A Simplified mRNA Development Workflow
Prepare and linearize plasmid DNA for transcription.
Generate RNA using T7 RNA polymerase and nucleotide substrates.
Introduce the desired 5′ cap using co-capping or enzymatic strategies.
Remove template DNA, enzymes, nucleotides and unwanted RNA species.
Prepare the RNA for cellular or in vivo delivery.
Evaluate dsRNA, residual enzymes, residual DNA, capping and other quality attributes.
1. DNA Template Preparation
In vitro transcription begins with a suitable DNA template. Available workflow components include plasmid extraction products and restriction enzymes for template preparation.
Endotoxin Free Plasmid Maxi Extraction Kit
BsaI
BspQI
2. In Vitro Transcription
Different transcription requirements may call for different enzyme and reagent configurations.
| Component | Available Options |
|---|---|
| T7 IVT Reagents | pUTP · N1-Me-pUTP · Low dsRNA · Thermostable |
| T7 RNA Polymerase | WT · Low dsRNA · Thermostable · Trace dsRNA |
| Supporting Enzymes | RNase Inhibitor · Inorganic Pyrophosphatase |
3. Capping Strategies
mRNA capping can be incorporated into the transcription workflow or performed through an enzymatic process.
Available raw materials include co-capping IVT reagents, Vaccinia Virus Capping Enzyme, mRNA Cap2′-O-Methyltransferase, S-Adenosylmethionine (SAM), and Cap1-related reagents.
4. Nucleotides and Modified Nucleotides
Researchers building their own IVT formulation can select individual nucleotide raw materials.
5. RNA Purification
Downstream processing may include template DNA digestion, removal of unwanted RNA species, and chromatographic RNA purification.
RNase R — linear RNA digestion for applicable circRNA workflows
Affinity Chromatography Purification Kit — Oligo dT-based purification
6. Transfection and Delivery
Delivery options include Fast LNP formulations designed for in vivo system delivery and in vitro primary-cell applications.
Making RNA Is Only Part of the Workflow
Quality-control tools are available for multiple process-related impurities and mRNA quality attributes.
| Quality Attribute | Assay |
|---|---|
| dsRNA | Double-Stranded RNA ELISA Kit |
| T7 Polymerase Residue | T7 RNA Polymerase ELISA Kit |
| Capping Efficiency | Sample Preparation Kit for Capping Efficiency Detection |
| Poly(A) Tail | Sample Preparation Kit for Poly(A) Tail Length Detection |
| Residual DNA | E. coli DNA / Plasmid Residual DNA Detection |
| Residual Process Enzymes | DNase I / Pyrophosphatase / RNase Inhibitor ELISA |
RNA Standards for Research and QC
Available standard materials include EGFP, Fluc, Cas9 and EPO mRNA, as well as saRNA and circRNA reference materials.
Building an mRNA Research Workflow?
Dana Bioscience can help source T7 RNA polymerases, IVT reagents, capping enzymes, modified nucleotides, RNA purification reagents, delivery products and mRNA quality-control assays for your research workflow.
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