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In Vitro Transcription

This In Vitro Transcription site is your portal for all things related to the synthesis of cRNA (copy RNA) from DNA in vitro. We provide you with all the background information, protocols for T7 T3 and S6 In Vitro Transcription, analysis steps, purification, visualization and troubleshooting information to become the ultimate expert for in vitro transcription!

In Vitro Transcription Table of Contents

What is In Vitro Transcription?

Transcription is the molecular process of the conversion of a DNA template into RNA molecules by RNA polymerase. In Vitro Transcription (in vitro meaning literally in glass) is transcription performed in a test tube, which uses purified components to essentially duplicate the process that occurs in vivo in a test tube. The in vitro system requires purified phage RNA polymerase, ribonucleotide triphosphates and linearized DNA template containing a phage RNA polymerase promoter site (see the diagram below for details).

In Vitro Transcription Diagram:

in vitro transcription

 

In Vitro Transcription Applications

The ability to synthesize RNA in the laboratory is critical to many techniques. Radiolabeled and non-radiolabeled RNA probes are required for many protocols, and they can be synthesized easily in small scale in vitro transcription reactions allowing their use in blot hybridizations and nuclease protection assays.

Such probes are much more sensitive than random-primed DNA probes.

Small scale reactions may also be used to synthesize RNA transcripts containing modified nucleotides for various biochemical and molecular biology studies. Large scale transcription reactions, generating up to 200 µg of RNA per reaction can be used for aRNA amplification, expression studies (microinjection, infection with viral transcripts, in vitro translation), structural analysis (protein-RNA binding), and mechanistic studies (ribozyme analyses). In this article, we present an overview of transcription, including requirements of in vitro transcription reactions and a comparison of conventional vs. large scale RNA synthesis.

 

Requirements for In Vitro Transcription ?

Systems are purified phage RNA polymerase, ribonucleotide triphosphates and a DNA template containing a phage RNA polymerase promoter sequence.

(either T7, T3, or S6)

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application.

 

Note however that if you use a T7 promoter site, you must use a T7 RNA phage polymerase.

RNA Phage Polymerases - T7, T3, or S6?

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application.

 

RNA Phage Polymerase Consensus Promoters

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application.

T7 T3 SP6 promoters

 

Template DNA for Run-off In Vitro Transcriptions

You need your target sequence inserted into a plasmid preferrably and digested for run-off transcription, or even a PCR product with T7 5' primers will do in this case.

 

If you use a PCR product, make sure there are at least 5 base pairs upstream of the T7 RNAP promoter. The polymerase needs something to bind to. It is a good idea to have a generic T7 promoter primer that you can use to PCR any template that has the promoter. The one I use has the sequence 5´-GAA ATT AAT ACG ACT CAC TAT A-3´ (promoter sequence in bold). This primer is also useful for sequencing plasmids that have the T7 RNAP promoter.

Note that if you are designing a template for transcription, T7 RNAP has certain base requirements for transcription initiation. If possible, the first two nucleotides after the promoter should be GG (to be transcribed; CC in the template strand), as these are preferred by T7 RNAP. The polymerase also works reasonably well with AG or GA as the starting nucleotides. If you need the 5´ end of your RNA to be something other than a purine, there are some post-transcriptional modifications that can be employed, typically involving ribozyme- or deoxyribozyme-mediated cleavage of your RNA. Maybe someone will put up a protocol for some of those strategies soon.

I generally recommend using 5–10 pmol of DNA template in a 100 μL transcription reaction. Does this mean you need to determine the concentration of your DNA? Not really, a reasonable estimate is good enough. For a 5000 base pair plasmid, 5 pmol is approximately 16 μg of DNA. For a PCR reaction, estimate the total number of pmols in your PCR by assuming that the reaction went to completion and half of your primers were used up (ex. a reaction with 50 pmol of each primer should yield approximately 25 pmol of extended product).

Buffers for In Vitro Transcription

Transcription Buffer Recipe:

for a solution of 1X transcription buffer add the following :

  • 50 mM Tris-HCl, pH 7.5
  • 15 mM MgCl2
  • 5 mM dithiothreitol (DTT)
  • 2 mM spermidine

It is recommended however to make 10X stock and store at -20°C.

 

10X NTP In Vitro Transcription Mix:

  • 20 mM ATP
  • 20 mM CTP
  • 20 mM GTP
  • 20 mM UTP

Store at -20°C.

 

Tips for In Vitro Transcription

  • Use RNAse inhibitors always!
  • Saving Money with T7 RNA Phage Polymerase: Clones with an N-terminal His-6 tag are available. If you obtain this clone, you can purify large amounts of T7 polymerase with high activity (ref: He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK. Protein Expr Purif. 1997, 9, 142–151).
  • To remove the DNA template, use an RNAse-free DNAse. We have used RQ1 DNase (Promega) added for 15 minutes and it works well. 

Storing RNA

  • RNA is best stored at a neutral pH with EDTA.
  • TE buffer is recommended (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), however this should be prepared with RNAse free purified (preferablly Millipore water).
  • RNase inhibitor is great for protecting your RNA.
  • Keep away from UV light or sunlight.
  • Store RNA at -20°C or -80°C.

Troubleshooting In Vitro Transcription Problems

Failed In Vitro Transcription Reactions

Causes include:
  • Poor-quality DNA template - Usually, DNA prepared using standard prep procedures is of sufficient quality however contaminants (e.g., ethanol or salts) can carry over and inhibit the reaction. Ethanol precipitation of the template is generally sufficient to resolve this problem.
  • Incorrectly linearized plasmid template: The sequence or restriction map of the template should be verified.
  • RNase contamination: RNase may be carried over from the plasmid purification procedure or inadvertently introduced by the investigator. RNasin® Ribonuclease Inhibitor should be included in all in vitro transcription reactions.
 

References on In Vitro Transcription

  1. Cazenave, C., Uhlenbeck, O.C. Proc. Natl. Acad. Sci. USA 1994, 91, 6972–6976.

References on Using PCR for In Vitro Transcription

  1. PCR Amplification Using Primers Containing the T7 Promoter Sequence Kain, K.C., Orlandi, P.A. and Lanar, D.E. (1991) Universal promoter for gene
    expression without cloning: Expression-PCR. BioTechniques 10, 366–74.
  2. van der Luijt, R. et al. (1994) Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC) gene by direct protein truncation test. Genomics 20, 1–4.

 

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