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cDNA Synthesis Troubleshooting


cDNA synthesis requires a clean RNA extraction and highly pure reagent. With RNA being very sensitive to degradation and prone to create secondary structures, cDNA cloning can be a frustrating experience. It is not uncommon to spend some time optimizing your protocol before getting the cDNA you need for your experiments.

If this is the case for you, have no fear! This ZAGENO troubleshooting guide is here to help.

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Low or No cDNA yield

  • Degraded RNA: Your RNA might have been degraded after the extraction. Check the integrity of your RNA with an agarose gel run
  • Short reverse transcription time: If you wish to amplify long RNA segments, check that your RNA is incubated with your retrotranscriptase for sufficient time
  • Secondary structure on the RNA: Increase the cDNA synthesis temperature
  • Wrong priming method: Switch from oligo(dT) to a random primer kit, or vice versa. Your application or reverse transcriptase might benefit from a mixture of the two
  • High dNTP concentration: Make sure that their final concentration is not above 0.5 mM
  • RNA contains inhibitors of cDNA synthesis: Use ethanol or LiCl precipitation to remove these impurities, or do a new extraction round

Amplified Product Larger Than Expected

  • Contamination in the RNA: The RNA extraction might be contaminated with genomic DNA. Make sure to treat your sample with DNase I

If you plan on repeating your experiment, make sure to include these controls to make troubleshooting easier

  • Use filter tips for your RNA extraction
  • Use an RNase decontaminating product to clean your work space before starting
  • Start your cDNA synthesis right after your RNA extraction
  • Check the quality of your RNA extraction before starting your cDNA synthesis