In vitro transcription describes the template-directed synthesis of RNA molecules; from short oligonucleotides to those of several kilobases. Synthetic RNA transcripts are therefore an important tool in structural, biochemical and genetic studies. Mistakes can lead to failed transcription or transcripts of a wrong size.
If this is the case for you, have no fear! ZAGENO's in vitro transcription troubleshooting guide is here to help.
- RNA template not pure: Contaminants like salts can inhibit the activity of the RNA polymerase. Desalt your template DNA by using a Clean-up kit
- Incorrect linearized template: Verify that the sequence and restriction map are correct and check an aliquot of purified DNA on an agarose gel
- RNase contamination from the plasmid purification step can degrade your RNA: Use an RNase inhibitor
- Inactive RNA polymerase: Always use a positive control template to ensure your in vitro transcription reaction works correctly
- Incorrect linearized template: Confirm the sequence and restriction sites and check an aliquot of purified DNA on an agarose gel
- Degradation of RNA sample buffer: Multiple freeze-thaw cycles as well as the use of old buffers can affect the reaction. The RNA will not run on its true size
- Nucleotide concentration is too low: The concentration should always be of at least 12 µM. Furthermore, adding “cold” rNTPs can increase the proportion of full-length transcripts
- The reaction might be terminating prematurely due to GC-rich templates: Decrease the temperature of the transcription reaction from 37 °C to 30 °C for full-length transcripts
Transcripts Longer than Expected
- Non-linearized plasmid in sample: After linearization of your template, check an aliquot on an agarose gel to confirm that the digestion was complete
- rUTP concentration is too high: Decrease the concentration of the nucleotide
- Templates with 3’ overhangs: Induce transcription from the opposite strand of DNA resulting in longer transcripts. Use restriction enzymes that produce 5’ overhangs or “blunt-ends” your DNA fragments with DNA Polymerase I
If you plan on repeating this experiment, follow these tips to make things easier
- Check your template DNA by agarose gel electrophoresis to make sure that your DNA template is linearized
- Use DNA templates of high quality
- Use the recommended nucleotide concentration
- Always use a positive control
Hopefully by following these in vitro transcription tips, your problems will soon become a thing of the past. If you're still having issues - or have extra tips - let us or the community know in the comments!