Introduction

Messenger RNA (mRNA) technology has transformed modern biotechnology, driving breakthroughs in gene therapy, vaccine development, and RNA-based therapeutics. At the heart of this innovation lies in vitro transcription (IVT) — a scalable, cell-free method for synthesizing high-quality mRNA from DNA templates.

Through controlled transcription reactions, IVT enables researchers to design, express, and deliver genetic instructions efficiently, making it an essential platform for both research and clinical applications. However, producing reliable mRNA requires more than transcription alone. Ensuring quality, purity, and consistency across every batch is critical for achieving reproducible biological performance and regulatory compliance.

The IVT Process in Custom mRNA Production

Custom mRNA production using IVT starts with a plasmid DNA template that includes a promoter, gene of interest, and a poly(A) tail. PackGene’s IVT workflow streamlines mRNA manufacturing — from DNA template to purified transcript — while maintaining scalability and cost-effectiveness across research-grade (RUO) and GMP-grade production.

This workflow ensures:

• Consistent template design for reproducible results
• Scalable production for preclinical and clinical applications
• Efficient transcription chemistry optimised for high yield and purity

Principles of IVT

IVT is a cell-free enzymatic process in which RNA polymerase synthesises RNA from a DNA template. The reaction requires:

• A DNA template with a promoter region
• RNA polymerase (e.g., T7, T3, SP6)
• Ribonucleotides (NTPs: ATP, CTP, GTP, UTP)
• Optimised transcription buffer

1. DNA Template Design

The DNA template provides the genetic blueprint for RNA synthesis. It contains the gene of interest and a promoter recognised by the RNA polymerase. During transcription, the enzyme reads the DNA in the 3′ → 5′ direction, producing RNA in the 5′ → 3′ direction.

A well-designed DNA template ensures:

• Accurate initiation at the correct site
• Proper transcription orientation
• High-fidelity RNA synthesis with minimal by-products

2. RNA Polymerase

RNA polymerases catalyse RNA synthesis by adding complementary ribonucleotides sequentially. T7, T3, and SP6 polymerases are commonly used, each recognising a specific promoter sequence.

3. Ribonucleotides (NTPs)

NTPs serve as the building blocks and energy source for RNA synthesis. Their concentration directly affects RNA yield, purity, and transcription efficiency. Balanced NTP levels are critical to producing high-quality mRNA suitable for downstream applications.

4. Transcription Buffer

The transcription buffer creates the optimal environment for RNA polymerase activity. It:

• Maintains pH stability
• Provides essential ions such as Mg²⁺ and K⁺
• Supports proper RNA folding and enzyme function

Fine-tuning the buffer improves both transcription efficiency and the integrity of the resulting RNA.

Structure of IVT mRNA

IVT mRNA, also called synthetic mRNA, is a single-stranded RNA molecule engineered to mimic natural mRNA. Its design includes five critical elements that regulate stability, translation efficiency, and protein expression:

Key Advantages of IVT for mRNA Production

Ensuring mRNA Quality with PackGene

Quality control (QC) is essential to guarantee reproducibility, safety, and efficacy. PackGene provides a focused, reliable QC package for every IVT mRNA batch, which includes:

• Appearance: Visual inspection for clarity and absence of particulates
• Purity: UV absorbance and size-based purity via capillary electrophoresis
• Concentration: Accurate quantification of mRNA yield
• Endotoxin Level: Gel Clot method to ensure safe thresholds for biological applications
• Integrity: Capillary electrophoresis confirms full-length, intact transcripts

For researchers requiring deeper analytical insights, optional add-on QC is available to assess residual DNA, dsRNA contamination, and host-cell impurities, providing flexibility based on project requirements.

This approach allows Southeast Asian researchers to choose QC depth appropriate to their application, from early-stage proof-of-concept studies to translational-grade mRNA manufacturing.

Accelerate Your mRNA Research with PackGene’s IVT Expertise

PackGene’s IVT service, supported by Atlantis Bioscience, offers a trusted, end-to-end solution for custom mRNA production. Their expertise in RNA synthesis, enzymatic processing, and purification ensures high-quality, transcriptionally active mRNA optimised for stability, purity, and translational performance.

Every standard IVT mRNA order includes core QC checks, delivering reliable and reproducible results, while optional add-on QC allows for greater analytical depth when required. Combined with Atlantis Bioscience’s regional support, this workflow provides Southeast Asian researchers with a complete solution — from design to delivery — enabling innovation to progress efficiently from concept to application.

References

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Kang DD, Li H, Dong Y. Advancements of in vitro transcribed mRNA (IVT mRNA) to enable translation into the clinics. Adv Drug Deliv Rev. 2023 Aug;199:114961. doi: 10.1016/j.addr.2023.114961.

Qin S, Tang X, Chen Y, Chen K, Fan N, Xiao W, Zheng Q, Li G, Teng Y, Wu M, Song X. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022 May 21;7(1):166. doi: 10.1038/s41392-022-01007-w.

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