In vitro transcription
The basic reaction: a single-subunit phage polymerase, its own 17–23 bp promoter, a linear template, four NTPs and magnesium. T7, SP6 and T3 each start only at their own promoter, which is what makes dual-promoter vectors useful, and what makes choosing the wrong enzyme produce a perfectly good RNA of the wrong sense. T7 is the most active and the most commonly used; SP6 gives somewhat lower yields and is often preferred for long transcripts.
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T7 / SP6 polymerase
The basic reaction: a single-subunit phage polymerase, its own 17–23 bp promoter, a linear template, four NTPs and magnesium. T7, SP6 and T3 each start only at their own promoter, which is what makes dual-promoter vectors useful, and what makes choosing the wrong enzyme produce a perfectly good RNA of the wrong sense. T7 is the most active and the most commonly used; SP6 gives somewhat lower yields and is often preferred for long transcripts.
How it differs from the standard method
- Each enzyme ignores the others' promoters entirely, so a vector with T7 one side and SP6 the other gives either strand on demand.
- T7 prefers to initiate with G and the first few transcribed bases influence yield, which is why promoter cassettes come with a defined +1 sequence.
- Reaction temperature is usually 37 °C; running at 30–32 °C lowers the yield slightly and reduces the double-stranded byproduct, which matters for RNA going into cells.
- Processivity is high, many kilobases, but long transcripts need lower nucleotide concentrations and more time rather than more enzyme.
- For a labelled probe, substituting part of one nucleotide with a labelled analogue sets the specific activity; too much substitution lowers both yield and hybridisation.
1A polymerase, its promoter, and an end to stop at
In vitro transcription is a single-subunit phage RNA polymerase (T7, SP6 or T3) copying a DNA template into RNA in a tube. The enzymes are remarkable for what they do not need: no transcription factors, no ATP-dependent initiation, no termination machinery.
The promoter
T7, SP6 and T3 each recognise only their own promoter, which also sets the transcription start site and the first transcribed bases. In a dual-promoter vector the two promoters face each other across the insert, so the enzyme you choose decides which strand you get.
The template end
A restriction site downstream of the insert, or the end of a PCR product. Nothing else stops the polymerase, so an incomplete digest means run-on transcripts.
Free magnesium
Nucleotides chelate Mg²⁺ one for one. At 7.5 mM each NTP there is 30 mM of chelator in the tube, so a reaction with standard 12 mM magnesium has almost no free Mg²⁺ left and simply stalls.
Nucleotide mass
The RNA cannot weigh more than the nucleotides that went in. 2 mM each NTP in 20 µL is about 50 µg of possible RNA; typical reactions convert half of it.
2The chemistry you have to balance
Two couplings make the reaction less forgiving than it looks. The first is magnesium.
- Phage RNA polymerase
- A single-subunit enzyme (T7, SP6, T3) that initiates at its own 17–23 bp promoter with no accessory factors and elongates processively for many kilobases.
- Run-on transcript
- RNA made past the intended 3′ end, because the template had no end there. The signature of a circular or incompletely linearised template.
- Abortive transcript
- Short products of a few to a few tens of nucleotides released during initiation. Normal, and removed by the clean-up.
- Free Mg²⁺
- Magnesium not bound to nucleotide. The quantity the polymerase actually responds to, and the reason NTP and Mg²⁺ concentrations cannot be chosen independently.
- Pyrophosphatase
- An enzyme that hydrolyses the pyrophosphate released at every nucleotide addition, preventing the magnesium pyrophosphate precipitate that stalls long high-yield reactions.
- Cap-0 / cap-1
- m7G added to the 5′ end (cap-0), and the same with 2′-O-methylation of the first transcribed nucleotide (cap-1). Cap-1 is what mammalian cells make and is markedly less immunogenic.
- Co-transcriptional capping
- Including a cap analogue in the reaction so that initiation starts with it. Requires the first transcribed bases to match the analogue, and costs some yield because the analogue competes with GTP.
- dsRNA byproduct
- Duplex RNA formed by self-primed 3′ extension and antisense transcription. The main innate-immune contaminant in in vitro transcribed RNA.
3Making RNA that a cell will treat as its own
An RNA destined to be translated needs three things the reaction does not provide by itself. A 5′ cap, because the initiation complex binds it and because a bare 5′ triphosphate is one of the structures the innate sensors evolved to recognise, so an uncapped transcript is both untranslated and inflammatory.
4How it fails
- No RNA, nothing obviously wrong. Usually free magnesium: a high-nucleotide reaction with a standard magnesium concentration. Check the arithmetic before changing anything else.
- A smear or a ladder instead of a band. A circular or incompletely linearised template, so the polymerase never stopped. Linearise completely, or transcribe from a PCR product.
- A low smear with no band at all. An RNase got in. Nuclease-free water, fresh tips, gloves, and a template that came out of a clean-up rather than a restriction digest.
- Good yield, no signal on the blot. The wrong polymerase on a dual-promoter vector, so the probe has the same sense as the target and cannot hybridise.
- A cloudy tube and a yield that stalled half-way. Magnesium pyrophosphate. Add pyrophosphatase, or shorten the reaction.
- Clean mRNA that is not translated and provokes an interferon response. Uncapped, untailed, unmodified or contaminated with double-stranded RNA, and because interferon shuts translation down, these problems amplify one another.
Key terms
Practise these as flashcards →Synthesis of RNA from a DNA template by a phage RNA polymerase in a defined buffer, outside a cell.
Cutting a plasmid downstream of the insert so the polymerase has an end to stop at. Must go to completion, and must be verified.
An RNA complementary to the target message, made by transcribing from the promoter that faces the insert from the appropriate side. The sense strand cannot hybridise.
Label per unit mass of probe, set by how much labelled nucleotide was included relative to its unlabelled counterpart.
A uridine analogue that replaces U entirely in therapeutic mRNA, lowering innate sensing and increasing translation.
Adding the 3′ tail, either encoded in the template or added enzymatically after transcription with E. coli poly(A) polymerase.
The fraction of transcripts that carry a cap. Measured, not assumed, usually by a cleavage assay and mass spectrometry or LC.
Transcription of a short single-guide RNA from a synthetic template, the cheapest route to guide RNA for CRISPR experiments, and one where run-on and dsRNA byproducts matter for cell viability.
When to use it
- Making a labelled antisense RNA probe for in situ hybridisation or a northern blot, where an RNA probe gives higher specific activity and more stable hybrids than a DNA one.
- Synthesising guide RNAs for CRISPR work, where a short synthetic template and a T7 reaction are far cheaper than chemical synthesis.
- Producing a defined RNA standard for absolute quantification by RT-qPCR or digital PCR, where a known number of transcript copies is the calibrant.
- Making mRNA for transfection, expression or vaccine work, which needs capping, tailing, modified nucleosides and removal of double-stranded byproduct.
- Generating substrate RNA for biochemistry: splicing assays, RNA-binding protein experiments, structural work or ribozyme studies.
Compared with alternatives
The main consumer of labelled antisense probes made this way. Prefer an RNA probe over a DNA oligo when sensitivity matters, because RNA:RNA hybrids are more stable and the probe can be made with high label density.
Also uses riboprobes from this reaction, and is where a sense-strand mistake shows up as a completely blank blot.
The opposite direction, RNA to DNA. In vitro transcribed RNA is often used as the spike-in or the absolute standard that lets a reverse transcription step be calibrated.
Guide RNAs can be transcribed rather than synthesised. Prefer synthetic, chemically modified guides for primary cells, where run-on products and dsRNA byproducts from a transcription reaction can be toxic.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
The basic reaction: a single-subunit phage polymerase, its own 17–23 bp promoter, a linear template, four NTPs and magnesium. T7, SP6 and T3 each start only at their own promoter, which is what makes dual-promoter vectors useful, and what makes choosing the wrong enzyme produce a perfectly good RNA of the wrong sense. T7 is the most active and the most commonly used; SP6 gives somewhat lower yields and is often preferred for long transcripts.
- Each enzyme ignores the others' promoters entirely, so a vector with T7 one side and SP6 the other gives either strand on demand.
- T7 prefers to initiate with G and the first few transcribed bases influence yield, which is why promoter cassettes come with a defined +1 sequence.
- Reaction temperature is usually 37 °C; running at 30–32 °C lowers the yield slightly and reduces the double-stranded byproduct, which matters for RNA going into cells.
- Processivity is high, many kilobases, but long transcripts need lower nucleotide concentrations and more time rather than more enzyme.
- For a labelled probe, substituting part of one nucleotide with a labelled analogue sets the specific activity; too much substitution lowers both yield and hybridisation.