In vitro synthesis of single-stranded RNA molecules is a widely used laboratory procedure that is critical to RNA research, as well as to RNA biopharmaceuticals. This technique is versatile in that it allows the researcher to tailor synthesis and introduce modifications to produce a transcript. Downstream applications include biochemical and molecular characterization of RNA for RNA-protein interactions and structural analyses, generation of RNA aptamers, synthesis of functional mRNAs for expression, and generation of small RNAs for alteration of gene expression (e.g., guide RNAs, RNAi). Furthermore, the use of in vitro synthesized RNA has been instrumental in the development of RNA vaccines and CRISPR/Cas9 genome editing tools, generation of pluripotent stem cells, as well as development of RNA amplification-based diagnostics.
High-yield robust IVT reactions require optimization of each reaction component. NEB offers six in vitro RNA synthesis kits, all of which have been optimized to generate reproducible yields of quality RNA. In addition, individual components can also be purchased for IVT and mRNA capping.
- Rio, D. C., et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220.
- Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington D.C.: ASM Press, 2007. 262-299.
- Jani B, Fuchs R. J Vis Exp. 2012 Mar 26 (61). PMID: 22473375
- Capped RNA Synthesis (E2040)
- Capping Protocol (M2080)
- DNA Template Preparation (E2040)
- Evaluation of Reaction Products (E2040)
- High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)
- Protocol for Dephosphorylation of 5'-ends of DNA using CIP (NEB #M0290)
- Purification of Synthesized RNA (E2040)
- RNA Synthesis with Modified Nucleotides (E2040)
- SP6 In Vitro Transcription Reaction Protocol (M0207)
- Standard RNA Synthesis (E2040)
- Protocol for Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)
- 2´-O-Methylation of Capped RNA (M0366)
- One-Step Capping and 2´-O-Methylation (M0366)
- Labeling Protocol (M2080)
- Capped RNA Synthesis (E2050)
- Evaluation of Reaction Products (E2050)
- Purification of Synthesized RNA (E2050)
- RNA Synthesis with Modified Nucleotides (E2050)
- Standard RNA Synthesis (E2050)
- Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)
- Protocol for Avoiding Rnase Contamination using Murine Rnase Inhibitor (M0314)
- Protocol for Avoiding Rnase Contamination using Rnase Inhibitor, Human Placenta (M0307)
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
- Evaluation of Reaction Products (E2060)
- mRNA Purification (E2060)
- mRNA Purification (E2065)
- mRNA Synthesis with Modified Nucleotides (E2060)
- mRNA Synthesis with Modified Nucleotides (E2065)
- Standard mRNA Synthesis (E2060)
- Standard mRNA Synthesis (E2065)
- Evaluation of Reaction Products (E2065)
- Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
- sgRNA Synthesis Using the HiScribe™ Quick T7 High Yield RNA Synthesis Kit (NEB #E2050)
- EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (E3322)
- Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
- Protocol for Dephosphorylation of 5´-ends of DNA using Quick Dephosphorylation Kit (M0508)
- Purification of Synthesized RNA (E2070)
- RNA Synthesis Protocols (E2070)
- Enzymatic PCR Cleanup Protocol
- Cappable-seq for prokaryotic transcription start site (TSS) determination
- Wu, M.Z., Asahara, H., Tzertzinis, G., Roy, B. (2020) Synthesis of low immunogenicity RNA with high-temperature in vitro transcription RNA; PubMedID: 31900329
- Eyler, D.E., Franco, M.K., Batool, Z., Wu, M.Z., Dubuke, M.L., Dobosz-Bartoszek, M., Jones, J.D., Polikanov, Y.S., Roy, B., Koutmou, K.S (2019) Pseudouridinylatio of mRNA coding sequences alters translation Proc Natl Acad Sci U S A; 116(46), 23068-23074.. PubMedID: 31672910, DOI: 10.1073/pnas.1821754116
- Potapov, V., Fu, X., Dai, N., Correa, I.R., Jr., Tanner, N.A., Ong, J.L (2018) Base modificatons affecting RNA polymerase and reverse transcriptase fidelity Nucleic Acids Res; 46(11): 5753-5763, PubMedID: 29750267
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This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene.