Mmessage mmachine rna transcription kit

Manufactured by Thermo Fisher Scientific

The MMessage mMachine RNA Transcription Kit is a product designed to facilitate in vitro transcription of RNA from DNA templates. The kit contains the necessary components, such as RNA polymerase, NTPs, and reaction buffers, to efficiently generate RNA transcripts from user-provided DNA templates.

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Lab products found in correlation

Collagenase type 1a, by Merck Group (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

MMessage mMachine kit MMessage mMachine Transcription Kits

3 protocols using mmessage mmachine rna transcription kit

Synthesis and Validation of 5-HT3A/3B cRNAs

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The cDNA clones of the human 5-HT_3A and 5-HT_3B subunits were provided by OriGen Inc. (Rockville, MD). Complementary RNAs (cRNAs) were synthesized in vitro using a mMessage mMachine RNA transcription kit (Ambion Inc., Austin, TX). The quality and size of synthesized cRNAs were confirmed by denatured RNA agarose gels.

Nebrisi E.E., Prytkova T., Lorke D.E., Howarth L., Alzaabi A.H., Yang K.H., Howarth F.C, & Oz M. (2020). Capsaicin Is a Negative Allosteric Modulator of the 5-HT3 Receptor. Frontiers in Pharmacology, 11, 1274.

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Expression of α4β2 Nicotinic Receptors

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Each subunit encoding mRNA was synthesized in vitro from linearized pGEMHE plasmid templates of Rattus norvegicus cDNA coding for α4 and β2 nAChR subunits using the mMessage mMachine RNA transcription kit (Ambion, Austin, TX). mRNA mixtures of α4 and β2 subunits were prepared at 2 μg:3 μg ratio, and 32.2 nL of this mixture was microinjected into each oocyte. The mRNA mixture was microinjected, using a displacement injector (Drummond Instruments, Broomhall, PA), into stage V and VI oocytes that had been extracted, incubated in collagenase Type 1A (Sigma, St. Louis, MO), and defolliculated by manual dissection. The injected oocytes were incubated at 19 °C for 3-4 days in ND-96 medium supplemented with albumin, gentamicin, tetracycline, and theophyline. Electrophysiological experiments were performed after the third or fourth day of mRNA injection.

Biaggi-Labiosa N.M., Avilés-Pagán E., Caballero-Rivera D., Báez-Pagán C, & Lasalde-Dominicci J.A. (2015). Engineering α4β2 nAChRs with reduced or increased nicotine sensitivity via selective disruption of consensus sites in the M3-M4 cytoplasmic loop of the α4 subunit. Neuropharmacology, 99, 273-284.

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Production of Replication-Defective Semliki Forest Virus Vectors

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All SV viral vectors used in these studies are replication defective. Vectors were produced as previously described. SV.IL12 plasmid used in this study was generated as published in 2002.⁸⁴ (link) SV empty is the same plasmid without an additional gene of interest (e.g., IL-12). SV.Luc was generated as described.²² (link) SV.GFP was generated as published in 2012.⁸⁵ (link) Briefly, plasmids carrying the replicon (e.g., SinRep-IL12) or DHBB helper RNAs were linearized with XhoI. In vitro transcription was performed using the mMESSAGE mMACHINE RNA transcription kit (Ambion). Helper and replicon RNAs were then electroporated into BHK cells and incubated at 37°C in α-MEM supplemented with 10% FCS. After 12 h, the media were replaced with Opti-MEM (Gibco-BRL) supplemented with CaCl₂ (100 mg/L) and cells were incubated at 37°C. After 24 h, the supernatant was collected, centrifuged to remove cellular debris, and frozen at −80°C. Vectors were titrated as previously described.⁸⁴ (link)

Scherwitzl I., Opp S., Hurtado A.M., Pampeno C., Loomis C., Kannan K., Yu M, & Meruelo D. (2020). Sindbis Virus with Anti-OX40 Overcomes the Immunosuppressive Tumor Microenvironment of Low-Immunogenic Tumors. Molecular Therapy Oncolytics, 17, 431-447.

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