T7 high yield rna transcription kit

Manufactured by Novoprotein

Sourced in China

The T7 High Yield RNA Transcription Kit is a laboratory tool designed for the in vitro transcription of RNA from DNA templates. The kit contains the necessary components, including the T7 RNA polymerase enzyme, nucleotides, and buffers, to facilitate the efficient synthesis of RNA molecules from a provided DNA template.

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Lab products found in correlation

Cap1 capping system, by Novoprotein (3 mentions) E coli poly a polymerase, by Novoprotein (2 mentions) Poly a tailing kit, by Thermo Fisher Scientific (1 mentions) Luciferin, by Yeasen (1 mentions) Hydrogenated soybean phosphatidylcholine hspc, by Lipoid (1 mentions) 1 1 dioctadecyl 3 3 3 3 tetramethylindotricarbocyaine iodide dir, by Yeasen (1 mentions) Rna clean beads, by Vazyme (1 mentions)

5 protocols using t7 high yield rna transcription kit

Preparation of cRNAs for Oocyte Microinjection

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To prepare cRNAs for human oocyte microinjection, constructs were linearized with the Not1 restriction enzyme, and the linearized products were purified by phenol (Tris-saturated):chloroform extraction and ethanol precipitation, and then dissolved in nuclease-free water. The purified DNA templates were transcribed in vitro using the T7 High Yield RNA Transcription Kit (Novoprotein, E131). Transcribed cRNAs were capped using the Cap1 Capping System (Novoprotein, M082) and poly(A) tails (~100 bp) were added using the E. coli Poly(A) Polymerase (Novoprotein, M012). cRNAs were purified by phenol(water-saturated):chloroform extraction and ethanol precipitated, and resuspended in nuclease-free water.
To prepare cRNAs for mouse oocyte microinjection, expression vectors were linearized and subjected to phenol (Tris-saturated):chloroform extraction and ethanol precipitation. The linearized DNAs were in vitro transcribed using the T7 message mMACHINE Kit (Invitrogen, AM1344). Transcribed mRNAs were added with poly(A) tails (~200–250 bp) using the Poly(A) Tailing Kit (Invitrogen, AM1350), recovered by lithium chloride precipitation, and resuspended in nuclease-free water.

Huang L., Li W., Dai X., Zhao S., Xu B., Wang F., Jin R.T., Luo L., Wu L., Jiang X., Cheng Y., Zou J., Xu C., Tong X., Fan H.Y., Zhao H, & Bao J. (2023). Biallelic variants in MAD2L1BP (p31comet) cause female infertility characterized by oocyte maturation arrest. eLife, 12, e85649.

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Luciferase-Based mRNA Vaccine Design

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The plasmid sequence was designed using Gaussia luciferase (GLuc) reporter gene as the open reading frame (ORF) whose signal peptide was replaced by tissue plasminogen activator (tPA) signal peptide, with the 5′UTR and 3′UTR derived from different genes, followed by the addition of the T7 promoter sequence before the 5′UTR (sequences showed in Supplementary Table S1). The plasmids were synthesized by GenScript (Nanjing, China) and cloned into pUC57 vector. mRNA was produced using T7 High-Yield RNA Transcription kit (Novoprotein, Shanghai, China) on linearized plasmids. Then, Cap 1 was added to the synthesized RNA by using the Cap 1 Capping System (Novoprotein, Shanghai, China) and Pseudo-UTP (Ψ-UTP, APExBIO, Houston, TX, USA) was fully substituted for UTP. The Poly(A) tails were added using E. coli Poly(A) Polymerase (Novoprotein, Shanghai, China).

Ding X., Zhou Y., He J., Zhao J, & Li J. (2024). Enhancement of SARS-CoV-2 mRNA Vaccine Efficacy through the Application of TMSB10 UTR for Superior Antigen Presentation and Immune Activation. Vaccines, 12(4), 432.

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Liposome Formulation and mRNA Synthesis

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Hydrogenated soybean phosphatidylcholine (HSPC) was purchased from Lipoid (Newark, USA). Cholesterol was from Nippon Fine Chemical (Osaka, Japan). Deuterated cholesterol was from Cambridge Isotope Laboratories (Tewksbury, USA). 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) was from A.V.T. (Shanghai, China). (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraeb-19-Y13-(4-methylpiperazin-1-Y1) propanoate (H3) was synthesized by BirdoTech (Shanghai, China) (6 ). EZ Cap™ firefly luciferase mRNA was provided by ApexBio (Houston, USA). 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) and luciferin were obtained from Yeasen (Shanghai, China). Water used through experiments was purified using a Millipore Milli-Q purification system (Bedford, MA). All other reagents were of chemical purity or analytical grade from Sangon Biotech (Shanghai, China).
For the synthesis of OVAL mRNA used in the in-situ hybridization (ISH) study, we used the T7 high yield RNA transcription kit from Novoprotein (Shanghai, China) and performed in vitro transcription (IVT) of a linearized DNA template from plasmid pCl-neo-sOVA (Addgene, Watertown, USA). The RNA was capped and attached poly(A) tails using enzymes included in the kit (Novoprotein, Shanghai, China).

Di J., Du Z., Wu K., Jin S., Wang X., Li T, & Xu Y. (2022). Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size. Pharmaceutical Research, 39(1), 105-114.

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CircRNA Production Protocol via IVT and RNase R

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CircRNAs were produced according to previous reports (7 (link)). In brief, the circRNA precursors were synthesized via IVT from the linearized circRNA plasmid templates with the T7 High Yield RNA Transcription Kit (Novoprotein Scientific Inc., China). After IVT, the RNA products were treated with DNase I for 35 min to digest the DNA templates. After DNase I digestion, GTP was added to the reaction at a final concentration of 2 mM, and then the reactions were incubated at 55°C for 20 min to catalyze the cyclization of circRNAs. Then, the RNA was column purified with RNA Clean Beads (Vazyme Biotech Co. Ltd., China). Then, the column-purified RNA was heated at 65°C for 5 min and cooled on ice. The reactions were treated with RNase R at 37°C for 30 min to further enrich the circRNAs. The RNase R-treated RNA was column purified. The sequences of circRNA-G and circRNA-RBD produced via Group I introns are provided in Tables S2 and S3.

Wan J., Wang Z., Wang L., Wu L., Zhang C., Zhou M., Fu Z.F, & Zhao L. (2023). Circular RNA vaccines with long-term lymph node-targeting delivery stability after lyophilization induce potent and persistent immune responses. mBio, 15(1), e01775-23.

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mRNA Synthesis for Protein Expression

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mRNA was transcribed in vitro using T7 high yield RNA transcription kit (Novoprotein) on linearized plasmids encoding dimeric RBDs. A 104 nucleotide-long poly(A) tail was transcribed into mRNA. UTP was replaced by 1-methylpseudourine-5′-triphosphate during in vitro transcription to generate nucleotide-modified mRNA. 5′-Capping was conducted using the Cap 1 capping system (Novoprotein). mRNA was purified by precipitation with LiCl at −20 °C overnight, centrifugation at 18,800 × g for 20 min at 4 °C, and resuspension with RNase-free water. Purified mRNA was verified by agarose gel electrophoresis and stored at −80 °C until use.

Chen Q., Du P., Han Y., Ma X., Zhang R., Rong X., Zhao X., Ma R., Yang H., Zheng A., Huang Q., Yan J., Wang H., Zhao X., Dai L., Gao G.F, & Wang Q. (2023). Rapid evaluation of heterologous chimeric RBD-dimer mRNA vaccine for currently-epidemic Omicron sub-variants as booster shot after inactivated vaccine. Biosafety and Health, 5(2), 89-100.

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