Takara Takara IVTpro T7 mRNA Synthesis

Takara IVTpro mRNA Synthesis Kit enables the production of large amounts of mRNA through in vitro transcription using an optimized, highly efficient T7 RNA polymerase. The kit includes separate NTPs for easy optimization or replacement with modified nucleosides, such as pseudouridine. The kit synthesizes up to 200 μg or more of mRNA per 20 μl reaction, includes DNase I to digest the template DNA after the in vitro transcription reaction and a lithium chloride (LiCl) solution to purify the synthesized mRNA for downstream applications.

The kit also includes a Positive Control Template (FLuc) plasmid containing the T7 promoter, transcription initiation sequence (AGG), 5- and 3-UTRs, FLuc CDS, and poly(A) sequence.

Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield.

Addition of the CleanCap Reagent AG (3’ OMe) in IVT reaction has no significant effect on mRNA yield(Panel A). HEK293T cells transfected with mRNA containing a cap structure show significatly higher protein expression compared to cells transfected with mRNA without a cap(Panel B).

The mRNA yield was proportional to the reaction volume up to 200 µl with no significant change in mRNA concentration(Panel A). Scaling up of the IVT reaction volume does not affect the quality of RNA product(Panel B).

Purification of RNA product using LiCl was comparable to purification with NucleoSpin column when two elutions were performed(Panel A). HEK293T cells transfected with mRNA purified using LiCl shows protein expression comparable to mRNA purified using NucleoSpin columns(Panel B).

The IVT reaction was performed using various sizes of DNA Templates with CleanCap Reagent AG (3 OMe) and N1-methyl pseudo UTP. Each resulting mRNA (200 ng) was denatured at 65° C for 10 min, followed by analysis using 1.2% formaldehyde gel electrophoresis.