In vitro synthesis of single-stranded RNA molecules is a routine laboratory procedure that is essential to the rapidly developing field of RNA research. This technique is versatile in that it allows the researcher to tailor synthesis, and introduce modifications that produce a transcript that is optimal for use in downstream applications. Though the ways in which RNA can be produced are countless, the same basic procedure is followed in all synthesis protocols.
The first step in in vitro RNA synthesis is to prepare the DNA template corresponding to the sequence of interest. To allow run off transcription, plasmid DNA template is generally linearized with a restriction enzyme. In addition to plasmid DNA, PCR products and synthetic oligonucleotides can be used as templates for transcription reactions. The template DNA is then transcribed by a T7, T3 or SP6 RNA phage polymerase in the presence of ribonucleoside triphosphates (rNTPs), (1-2). The polymerase traverses the template strand and uses base pairing with the DNA to synthesize a complementary RNA strand (using uracil in the place of thymine). The RNA polymerase travels from the 3′ – 5′ end of the DNA template strand, to produce an RNA molecule in the 5′ – 3′ direction (2).
- 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
Protocols for In vitro Synthesis & Processing
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- 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® Electroporation
- EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (NEB #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)
- Labeling Protocol (M2080)
- mRNA Purification (E2060)
- mRNA Purification (E2065)
- mRNA Synthesis with Modified Nucleotides (E2060)
- mRNA Synthesis with Modified Nucleotides (E2065)
- 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 (NEB #M0386)
Reagents for RNA Sample Preparation
RNA Polymerase Selection Chart
RNA Ligase Selection Chart
cDNA Synthesis Selection Chart
DNA Ligase Selection Chart
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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.