mRNA Synthesis by In Vitro Transcription
VectorBuilder offers in vitro transcription-based mRNA synthesis services for custom mRNAs of up to more than 10 kb in length and suitable for a variety of downstream applications including in vitro translation, biochemical studies, protein expression after injection into cells or embryos, and vaccine development research.
Our mRNA synthesis process is highly optimized for producing high-quality mRNAs utilizing our proprietary in vitro transcription vectors that are used in our vector cloning services. We can also synthesize mRNAs from customer-supplied vectors. While our research-grade mRNA synthesis services are suitable for early-stage developmental studies, we also offer GMP-grade mRNA synthesis services for clinical applications. In addition to mRNAs, we can generate small RNAs including gRNAs for CRISPR/Cas9 targeting and other small RNAs for applications requiring short RNAs of defined sequences.
Types of RNAs offered
- mRNAs of up to >10 kb in length
- gRNAs for CRISPR applications
- Other short RNAs of defined sequences (microRNA, siRNA etc.)
- Utilizes the highly efficient and versatile in vitro transcription approach based on the bacteriophage derived T7 RNA polymerase (RNAP)
- 5’ m7G-cap and 3’ polyA tails added to transcripts for enhancing mRNA stability and facilitating efficient protein translation
- Ability to generate mRNAs with 110 bp long polyA tails by the incorporation of polyA tracts into the template plasmid
- Variety of RNA modification options including addition of modified nucleotides to modulate innate immune response
- Post-transcriptional DNase and phosphatase treatments to facilitate the removal of DNA template and terminal 5’ triphosphates, respectively
- Variety of RNA purification options including silica gel purification and magnetic bead-based purification
Price and turnaround
|>500 ng/ul||Molecular biology, cell culture, and in vivo use||25 ul, sterile||$399*||2-4 days|
|50-200 ug||100 ul, sterile||Please inquire||Please inquire|
|0.25-1 mg||500 ul, sterile|
* Please note that the price shown in the table above applies for mRNA production only and does not include the cost of vector cloning. For mRNA transcription service, 5’ m7G-cap and 3’ polyA tails are added by default, but additional fee may apply if requesting other RNA modification. While cap 0 is added to mRNAs by default, mRNAs with cap 1 can be provided upon request. Please send us a design request to inquire about available RNA modification options.
If customer-supplied vectors are needed, please submit your materials information on "Support" > "Materials Submission Form". Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur an additional QC charge starting from $120 per item, depending on the type and usage of item. Please note that production cannot be initiated until customer-supplied materials pass QC.
Shipping and storage
Our mRNA is stored in either 1mM sodium citrate (pH 6.4) or nuclease-free water depending on the application and shipped on dry ice. Upon receiving, mRNA samples should be stored at -80°C for long term (stable for at least 6 months), or -20°C for use within one week. The shelf life for mRNA is approximately one year. Please avoid repeated freeze-thaw cycles of mRNA since it can cause significant RNA degradation.
mRNA production process and QC
For mRNA production, we use the in vitro transcription approach which utilizes the T7 promoter upstream of a DNA template sequence of interest to facilitate highly efficient production of mRNA by the bacteriophage T7 RNAP, under appropriate reaction conditions and in the presence of nucleotide triphosphates. The T7 RNAP is a highly robust enzyme and transcription reactions using this enzyme can produce large amounts of functional RNA within a few hours. Our typical mRNA production workflow starts with designing and synthesis of the template DNA sequence followed by its cloning into an in vitro transcription vector. The plasmid DNA is then purified, validated, and linearized before being subsequently subjected to the in vitro transcription reaction which results in the generation of the desired transcript. The mRNA is then purified by silica gel purification which is our default purification process. We can also purify mRNAs by magnetic-bead purification upon request.
Figure 1. Typical workflow of mRNA synthesis by in vitro transcription.
All mRNAs produced by VectorBuilder undergo stringent quality control to ensure that they are free of contaminants and contain the correct sequence exactly as designed. This involves: 1) validation of the in vitro transcription vector containing the cloned DNA template sequence by restriction digestion analysis and Sanger sequencing; 2) final QC of the synthesized RNAs by denaturing PAGE or agarose gel electrophoresis depending on the size of the transcript.
Our in vitro transcription-based mRNA production process has been highly optimized to enhance mRNA stability and facilitate efficient protein translation. An example of successful protein translation from mRNA produced using our in vitro transcription vector is shown in Figure 2 below.
Figure 2. 293T cells were transfected with EGFP mRNA carrying a 5' cap 1 structure. Images were taken at 48 hours post-transfection. Magnification: 100x. Left: bright field. Right: EGFP.
How to Order
Order both vector cloning and RNA preparation
Order RNA preparation from your own vector
What are the advantages of in vitro transcription-based RNA synthesis over chemical synthesis of RNA?
Producing RNAs by in vitro transcription offers several advantages over chemical synthesis which include:
1) Cost-effectiveness and technical simplicity – Chemical synthesis of RNA is typically performed by automated solid-phase oligonucleotide synthesis utilizing phosphoramidite chemistry, which involves several cycles of specific synthesis steps making the process technically complex and expensive. In vitro transcription-based RNA synthesis on the other hand is a highly versatile, yet simple and cost-effective technique routinely used by many laboratories to generate a variety of transcripts.
2) Higher yields – In vitro transcription based-RNA synthesis typically utilizes the bacteriophage derived T7 RNAP for generating desired RNA transcripts from DNA template sequences located downstream of a T7 promoter. The T7 RNAP is a robust enzyme with significantly high processivity and frequency, allowing milligrams of mRNA to be synthesized from very small reaction volumes, thereby making this process highly scalable.
3) Flexibility - In vitro transcription allows synthesis of RNAs ranging in length from a few hundred nucleotides to more than 10 kilobases which can be used for a variety of downstream applications, whereas chemical synthesis allows RNAs of only up to 200 bases to be synthesized, with longer sequences being highly prone to errors.
What makes mRNAs attractive candidates for vaccine therapy?
mRNAs have recently emerged as promising candidates in the field of vaccine development as they offer several advantages over traditional virus or DNA-based vaccines including:
1) Efficacy - mRNA vaccines have been shown to be highly effective in mounting reliable immune response without any significant side effects.
2) Ease of delivery - Formulating mRNA vaccines into carrier molecules allows them to be efficiently delivered into host cells, followed by their rapid uptake and expression in the cytoplasm without having to cross the nuclear membrane. Moreover, they can be easily administered via various routes including subcutaneous, intranodal, intradermal, intramuscular or intravenous injections which play a critical role in determining the how safe and effective the vaccine will be.
3) Safety - mRNA vaccines are non-infectious and non-integrating without any associated risks of insertional mutagenesis which makes them extremely safe compared to viral vector-based vaccines. Additionally, their safety profile can be further improved by the incorporation of modified nucleotides that can modulate innate immune response upon delivery to host cells.
4) Production ease and scalability - mRNA vaccines can be produced very rapidly at relatively low costs and at large scales utilizing the in vitro transcription approach which is technically far less challenging than producing virus-based vaccines. Moreover, since mRNA vaccines allow antigens to be synthesized in situ, this eliminates the need for protein purification and stabilization which can be hard to achieve for certain antigens. The relative ease of production enables mRNA vaccine candidates to be rapidly translated from the early discovery phase to the clinical and commercial phases in response to epidemics.
5) Versatility - mRNA offers a highly versatile platform for vaccine development since it can be used for simultaneous expression of multiple viral antigens using a single vaccine, which is typically harder to achieve with other conventional vaccine types.