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 five 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
FAQs for In vitro Synthesis (IVT)
Protocols for In vitro Synthesis (IVT)
- 2´-O-Methylation of Capped RNA (M0366)
- Cappable-seq for prokaryotic transcription start site (TSS) determination
- Capped RNA Synthesis (E2040)
- Capped RNA Synthesis (E2050)
- Capping Protocol (M2080)
- DNA Template Preparation (E2040)
- Electroporation of Cas9 RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporator
- EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (E3322)
- Enzymatic PCR Cleanup Protocol
- Evaluation of Reaction Products (E2040)
- Evaluation of Reaction Products (E2050)
- Evaluation of Reaction Products (E2060)
- Evaluation of Reaction Products (E2065)
- High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- Labeling Protocol (M2080)
- mRNA Purification (E2060)
- mRNA Purification (E2065)
- mRNA Synthesis with Modified Nucleotides (E2060)
- mRNA Synthesis with Modified Nucleotides (E2065)
- One-Step Capping and 2´-O-Methylation (M0366)
- Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
- Protocol for Avoiding Rnase Contamination using Murine Rnase Inhibitor (M0314)
- Protocol for Avoiding Rnase Contamination using Rnase Inhibitor, Human Placenta (M0307)
- Protocol for Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)
- Protocol for Dephosphorylation of 5´-ends of DNA using Quick Dephosphorlyation Kit (M0508)
- Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)
- Protocol for Dephosphorylation of 5'-ends of DNA using CIP (NEB #M0290)
- Purification of Synthesized RNA (E2040)
- Purification of Synthesized RNA (E2050)
- Purification of Synthesized RNA (E2070)
- RNA Synthesis Protocols (E2070)
- RNA Synthesis with Modified Nucleotides (E2040)
- RNA Synthesis with Modified Nucleotides (E2050)
- sgRNA Synthesis Using the HiScribe™ Quick T7 High Yield RNA Synthesis Kit (NEB #E2050)
- SP6 In Vitro Transcription Reaction Protocol (M0207)
- Standard mRNA Synthesis (E2060)
- Standard mRNA Synthesis (E2065)
- Standard RNA Synthesis (E2040)
- Standard RNA Synthesis (E2050)
- Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
- Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
Minding your caps and tails
Applications of synthetic mRNA have grown and become considerably diversified in recent years.
Latest NEB Expressions
This edition of NEB expressions discusses breakthroughs in Golden Gate Assembly research, that now enables 20+ fragment assembly with high efficiency and accuracy. New products include the NEBNext® Single Cell/Low Input RNA Library Prep Kit and the NEBNext Direct® Custom Ready Panels for NGS Target Enrichment. Lastly, learn about the importance of basic research at NEB in an interview with NEB’s Research Director.
RNA Synthesis Brochure
Learn more about NEB's products for RNA synthesis, which range from template generation to poly(A) tailing.
RNA Technical Guide
Find in depth information on tools designed to streamline your RNA workflows, including RNA extraction & purification, qPCR and RT-qPCR, RNA-seq, RNA synthesis and gRNA synthesis.
- Avoiding Ribonuclease Contamination
Other Tools & Resources
Recommended HiScribe RNA Synthesis Kits by Application
RNA Polymerase Selection Chart
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene.