A widely used molecular biology technique, in vitro transcription uses bacteriophage DNA-dependent RNA polymerases to synthesize template-directed RNA molecules. Enzymes like bacteriophage SP6, T3 and T7 RNA polymerases are used to produce synthetic RNA transcripts, which can be used as hybridization probes, as templates for in vitro translation applications, or in structural studies (X-ray crystallography and NMR). Synthesized RNA transcripts are also used for studying cellular RNA functionality in processes such as splicing, RNA processing, intracellular transport, viral infectivity and translation.
Problems in the transcription reaction can result in complete failure (i.e., no transcript generated) or in transcripts that are the incorrect size (i.e., shorter or longer than expected). Below is a discussion of the most common causes of in vitro transcription problems.
Complete failure of the in vitro transcription reaction might be the result of using a poor-quality DNA template. DNA prepared with standard miniprep procedures should be of a sufficient quality for in vitro transcription. However, contaminants such as ethanol or salts carried over from the DNA purification process can inhibit the RNA polymerases. Typically, precipitating the DNA template with ethanol and resuspending it will resolve the contamination issues.
Transcription templates that have been linearized incorrectly can result in transcription failure. If you are using a linearized template for transcription, verify that the sequence and restriction map are correct.
Finally, if you are confident of the quality of your template DNA and are still failing to see any transcription product, RNase contamination might be the culprit. RNase can be carried over from the plasmid purification process or inadvertently introduced by inappropriate handling. RNase will degrade and RNA it comes in contact with. To prevent RNase from destroying your transcripts, use an RNase inhibitor such as RNasin® Ribonuclease Inhibitor in your transcription reactions. You can learn more about working with RNA and creating an RNase-free environment in this article.
If you are not getting full-length transcripts, there are a number of different things to investigate. If you are using a linearized template, confirm the sequence and restriction sites. Unexpected cut sites can mean that the transcription template is shorter than expected. Try linearizing your template plasmid with a different enzyme.
The presence of cryptic phage RNA polymerase termination sites could be causing the transcription reaction to stop before the end of template sequence. To resolve this, subclone your template into a different plasmid with a different RNA polymerase promoter.
Adjusting the transcription reaction conditions can resolve the problem of incomplete transcription. If the nucleotide concentration is too low, it might be limiting the reaction. Always use a nucleotide concentration of at least 12µM, and if you think this is the problem, you can increase the concentration to 20–50µM (Note: If you are synthesizing labeled transcripts, increasing the nucleotide concentration may require adding cold NTP, and this will decrease the specific activity of the final product).
If the template sequence is G/C rich, the transcription reaction might be terminating prematurely. For these templates, the amount of full-length transcript can be increased by decreasing the temperature of the transcription reaction (1).
Transcripts Longer than Expected
Transcripts that are longer than expected can result when the template plasmid has not been completely linearized. After linearizing your template, check an aliquot on an agarose gel to confirm that the digestion was complete.
Templates with 3’ overhangs can cause the phage RNA polymerase to extend on the opposite strand of the linearized template, resulting in longer transcripts containing the sequence of the complimentary strand. You can avoid this by using restriction enzymes that produce 5’ overhangs or blunt ended fragments (2, 3).
It is frustrating when a reaction fails or gives unexpected results. Keeping these common causes of problems in mind as you plan your in vitro transcription reactions could help you avoid frustrating results.
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- Krieg, P. and Melton, D. (1987) Meth. Enzymol. 155, 397.
- Triana-Alonso et al. (1995) J. Biol. Chem. 270, 6298–307.
- Schenborn and Mierendorf (1985) Nucl. Acids Res. 13, 6223–36.