Takara IVTpro mRNA synthesis FAQs

Yes, the Cloning Kit for mRNA Template is also available as a standalone kit.

The only material you need to generate a DNA template for in vitro transcription is a target coding sequence (also called gene of interest or GOI). Note that the linearized template vector in Cloning Kit for mRNA Template carries the following sequences in this order:

• T7 promoter sequence
• Transcription start sequence (AGG) for CleanCap AG or other compatible cap analogs
• 5′ UTR, including a Kozak sequence
• 3′ UTR, Poly(A) sequence
• Multiple restriction enzyme sites (e.g., HindIII or BspQ I)

An entire sequence data (.gb) of the linearized template vector is available under ‘Vector Documents’ on the Takara IVTpro mRNA synthesis system’s product page. We recommend including a stop codon in a target sequence to avoid stop codon readthrough.

To linearize the vector with Cloning Kit for mRNA Template, we recommend using an appropriate restriction enzyme, either HindIII or BspQ I, which has a digestion site located right after the Poly(A) sequence. Note that if your GOI and/or junction sites between the GOI and vector sequence contain the restriction site(s), the site(s) need to be removed by altering the codon(s) without changing the amino acid sequence. Other restriction sites are also available but not recommended because extra sequences after poly(A) tail are known to affect mRNA translation.

After restriction enzyme digestion, it is better for the linearized DNA to have 5′ overhangs or blunt ends. It is best to avoid 3' protruding ends, as these may produce undesirable RNA sequences.

CleanCap^® Reagent AG (TriLink BioTechnologies, No. N-7113 or N-7413) is recommended because the linearized template vector in Cloning Kit for mRNA Template has a transcription start site AGG as a default sequence. ARCA and conventional m7G cap analogs can be also used. However, efficient IVT reaction with these cap analogs requires the transcription start site (+1 position) to start with a G nucleotide. Therefore, you must modify the transcription start sequence from AGG to GGG. Please refer to this technical note or the User Manual for detail.

We have confirmed that a GOI as large as ~12 kb could be successfully constructed using the linearized template vector in Cloning Kit for mRNA Template. Please note that the yield and quality of transcripts may be compromised for fragments larger than 4 kb. We confirmed that FLuc mRNA (approximately 1.9 kb) was generated with high quality and high yield using our kits.

Takara IVTpro mRNA Synthesis System includes a cloning kit for preparing the DNA template, which is also sold separately: Cloning Kit for mRNA Template. If you already have a DNA template for in vitro transcription, we recommend using the standalone kit, Takara IVTpro T7 mRNA Synthesis Kit.

You can use a linearized DNA vector included in Cloning Kit for mRNA Template or double-stranded DNA template that contains a T7 promoter sequence (i.e., a PCR amplicon or fully synthesized DNA fragment).

Yes. Both the 5’ and 3’ structures are critical for efficient target translation and mRNA stability in cells. Missing either of the structures will result in unsatisfactory translation and protein expression. For DNA template preparation, we recommend using Cloning Kit for mRNA Template, contained in the Takara IVTpro mRNA Synthesis System. This cloning kit is also sold separately. The T7 vector in this cloning kit contains a transcription start site that is compatible with CleanCap® AG, UTRs, and poly(A) sequence.

RNA yield of FLuc mRNA (approx. 1.9 kb size) is about 200 μg/reaction. If desired, the IVT reaction volume can be scaled up 10-fold (up to 200 μl reaction volume) without compromising the yield.

10X Enzyme Mix contains T7 RNA Polymerase ver. 2 (Cat. #2541A) plus a proprietary mixture of supplemental enzymes. Therefore, the T7 promoter sequence in DNA templates is essential for in vitro transcription reaction.

If resulting mRNA require cap structures, we recommend either separately purchasing your own cap analogs, such as CleanCap Reagent AG (TriLink BioTechnologies, No. N-7113 or N-7413), or using enzymatic post-transcriptional capping systems, including Vaccinia Capping Enzyme (Cat. #2460) or Faustovirus Capping Enzyme (Cat. #2480) along with mRNA Cap 2′-O-Methyltransferase (Cat. #2470). Please note that neither Takara IVTpro mRNA Synthesis System nor Takara IVTpro T7 mRNA Synthesis Kit contains capping systems.

Yes. Modified NTPs, such as pseudo-UTP, etc., can be used instead of UTP to reduce the innate immune response. When using modified NTPs, replace the corresponding NTP with an equivalent amount. Please refer to the User Manual for details.

Both the Takara IVTpro mRNA Synthesis System and the Takara IVTpro T7 mRNA Synthesis Kit contain LiCl for RNA purification. If your RNA size is less than 300 bp or if the RNA concentration is less than 0.1 μg/μl, we recommend purifying the RNA by spin column (NucleoSpin RNA Clean-up, Cat. #740948.10/.50/.250) or by traditional ethanol precipitation after phenol-chloroform extraction.

The 5’ cap structure can be added co-transcriptionally using cap analogs (such as CleanCap, ARCA, or mCap) or post-transcriptionally using Vaccinia Capping Enzyme (Cat. #2460) or Faustovirus Capping Enzyme (Cat. #2480). Please refer to detailed information about 5′ capping of mRNA in our mRNA synthesis learning center.

A protocol for the co-transcriptional capping method can be found in the User Manual for the Takara IVTpro mRNA Synthesis System or the Takara IVTpro T7 mRNA Synthesis Kit. These are located in the Documents tab of the product page.

For a protocol of the post-transcriptional capping method, please refer to the User Manual for Vaccinia Capping Enzyme (Cat. #2460) or Faustovirus Capping Enzyme (Cat. #2480). Additionally, the cap-1 structure can be created using mRNA Cap 2′-O-Methyltransferase (Cat. #2470).

No, our Vaccinia Capping Enzyme (Cat. #2460) does not have thermostable capability. Faustovirus Capping Enzyme (FCE, Cat. #2480) exhibits high enzyme activity over a broader temperature range, making it suitable for various 5′ sequences. 

Capping Enzymes—Vaccinia Capping Enzyme (Cat. #2460) or Faustovirus Capping Enzyme (Cat. #2480)—can modify and convert an uncapped RNA (both 5′-triphosphate and 5′-diphosphate RNA) into m7G-capped RNA (cap-0 RNA structure).

The capping efficiency by Capping Enzymes is expected to be close to 100% (data not shown). Although the procedure can be time-consuming, high capping efficiency is a major advantage over traditional co-transcriptional methods, including ARCA.

The m7G-capped structure (cap 0) is required for 2′-O-methylation on the first nucleotide. In addition, 3'-O-methylated m7G-cap, such as ARCA, can also be used as a substrate for mRNA Cap 2′-O-Methyltransferase (Cat. #2470).

Two options, either the 1-step or 2-step methods, are available for preparing cap-1 RNA from uncapped RNA. In the 1-step, direct modification method, Vaccinia Capping Enzyme (Cat. #2460) or Faustovirus Capping Enzyme (Cat. #2480) will generate a cap-0 structure while mRNA Cap 2′-O-Methyltransferase (Cat. #2470) simultaneously generates a cap-1 structure within the same tube. In the 2-step, indirect modification method, these two enzymatic reactions will occur sequentially as a separate step. The 2-step method needs at least one RNA purification step between the reactions. For more details, please see this technical note on post-transcriptional capping. 

No. The mRNA Cap 2′-O-Methyltransferase (Cat. #2470) can convert a cap-0 into cap-1 RNA structure, but it is not possible to further convert to cap-2 RNA structure.

If the full RNA length is less than 200 bp, we recommend extending the incubation time with capping enzyme from 30 min to 1 hr for cap-0 RNA preparation. For cap-1 RNA preparation using both a capping enzyme and mRNA Cap 2′-O-Methyltransferase (Cat. #2470), extending the incubation time from 1 hr to 2 hr will enhance the capping and conversion efficiency.

Yes. Please refer to our selection guide to choose the right product(s) for your needs.