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In vitro transcription (IVT) is the cell-free synthesis of RNA: a bacteriophage RNA polymerase such as T7 copies a DNA template that carries the matching promoter, using nucleotide triphosphates in a buffered, RNase-free reaction. The tube yields RNA you can use as a probe, a transfection reagent or an mRNA, but the product is a mixture that usually needs clean-up before use. This page is general background for researchers and lab staff, not a bench protocol.
Short answer
IVT is how labs synthesize RNA outside a cell. A double-stranded DNA template with a phage promoter is incubated with the polymerase and four nucleotides, and the enzyme writes RNA from the promoter to the end of the template. Template design and post-reaction clean-up decide most of the quality of the result.
Definition and scope
IVT uses a purified RNA polymerase, a DNA template, nucleotides and a reaction buffer instead of a living cell. T7 RNA polymerase, encoded by the T7 bacteriophage, is described as the best characterized member of the widespread family of RNA polymerases, and the mRNA vaccines approved by the FDA to date have been transcribed in vitro by T7 RNA polymerase (Molecules 2024, PMC11477551). Typical uses listed in a vendor T7 kit manual include microinjection, transfection, in vitro translation, structure and function studies, in situ hybridization probes and Northern blots (Jena Bioscience RNT-101 manual, version 0007, May 2026). Labelled or modified nucleotides (biotin, fluorescent) and cap analogs can be built into the same reaction; capping is covered separately in mRNA capping.
How it works in practice
The template
The template is linearized plasmid DNA or a PCR product containing a double-stranded T7 class II (phi2.5) or class III (phi6.5) promoter upstream of the target sequence. The same manual states that template quality directly influences yield and quality. A linearized plasmid should be fully digested and free of RNase, protein and salts, and blunt or 5′ overhang ends are recommended. A PCR mixture can be used directly, but a purified PCR product usually gives better yield. If the goal is an mRNA, the template must encode the UTRs, be in the correct orientation and include a poly(A) tail, or the poly(A) tail can be added afterwards with poly(A) polymerase.
The reaction
The manual lists the components as buffer, DTT, four NTPs, template and a polymerase mix containing RNase inhibitor. It calls RNase-free working (gloves, RNase-free tubes and tips) critical even when an RNase inhibitor is present. For background on RNase-inactivated water, see DEPC. In that vendor’s example, a 20 µL reaction with 1 µg of a 1.4 kb control template gave roughly 140 to 160 µg RNA after 30 minutes; the manual itself notes that yield varies with promoter design, length and secondary structure, so treat the figure as one kit’s specification, not a general expectation.
Promoter variants and related concepts
| Feature | Class III (phi6.5) | Class II (phi2.5) |
|---|---|---|
| Minimum promoter (5′ to 3′) | TAATACGACTCACTATAGNN | TAATACGACTCACTATTANN |
| First base incorporated into the RNA | G | A |
| Preferred NN | CG | GG |
Source: Jena Bioscience RNT-101 manual, version 0007. The first transcribed base therefore follows from the promoter you choose, which matters when the 5′ end has to match a design. IVT is also distinct from amplification methods: for a different cell-free nucleic-acid workflow see isothermal amplification, and for protein made inside cells see protein expression in E. coli.
Where it matters: what comes out of the tube
The reaction product is not only the intended transcript. The table maps each byproduct to the source-stated concern and the clean-up it points to.
| Component in the product | Why it matters (as stated by the source) | Source-stated handling |
|---|---|---|
| Double-stranded RNA (dsRNA) | Key immunogenic byproduct of IVT mRNA | Complex downstream column purification is the traditional removal method (Molecules 2024) |
| Fragmented mRNA | Largest proportion of impurities in the reaction, linked to Mg2+-accelerated hydrolysis and premature termination (Molecules 2024) | Addressed through reaction optimization in the cited paper (Molecules 2024) |
| 5′-triphosphate on the RNA | Known to trigger a RIG-I mediated innate immune response in mammalian cells (Jena manual, citing Wienert 2018 and Kim 2018) | Phosphatase (CIP) treatment is recommended for transfection use |
| Residual DNA template | May need removal depending on the application | Salt-resistant DNase treatment |
| Proteins, salts, unincorporated nucleotides | Carried over from the reaction | Spin-column clean-up (the manual says LiCl precipitation may work but is untested) |
RNA concentration is quantified by A260, where an absorbance of 1 corresponds to about 40 µg/mL single-stranded RNA (Jena manual).
Common misunderstandings
- “The tube contains pure target RNA.” It also contains template DNA, enzymes, salts, free nucleotides and side products such as dsRNA and fragments.
- “Any RNA from IVT is safe to transfect.” The 5′-triphosphate can trigger RIG-I signalling, which is why the vendor manual recommends phosphatase treatment for transfection probes.
- “A published yield applies to my template.” The vendor states yield depends on promoter design, length and secondary structure.
- “An RNase inhibitor replaces clean technique.” The manual calls RNase-free handling critical even with an inhibitor in the mix.
FAQ
What does it mean to synthesize RNA in vitro?
It means making RNA in a tube from a DNA template with a purified polymerase, rather than isolating RNA from cells.
Which polymerase is used?
T7 RNA polymerase is the best characterized and is the enzyme behind the FDA-approved mRNA vaccines to date (Molecules 2024); the T7 kit manual cited here describes T7 class II and class III promoters.
Does IVT make capped, tailed mRNA by itself?
Not automatically. The template must encode the UTRs and a poly(A) tail, or a tail is added enzymatically; capping is a separate design choice (see mRNA capping).
Sources
- Jena Bioscience, HighYield T7 RNA Synthesis Kit (RNT-101) instructions, version 0007, updated 15 May 2026: jenabioscience.com (read 2026-11-08). Kit-specific figures are attributed to that vendor.
- Molecules 2024, “Effective Synthesis of mRNA during In Vitro Transcription with Fewer Impurities Produced”: PMC11477551 (read 2026-11-08).
