cDNA synthesis relies on clean RNA extraction and handling beforehand, as RNA is very sensitive to degradation and prone to create secondary structures.
As a result, cDNA cloning can be a frustrating experience and it is not uncommon to spend substantial time optimizing your protocol before getting the cDNA you need for your experiments.
Below we list some of the common problems you may encounter, their cause, and how to solve them.
Problem: Low or No cDNA yield
Possible Cause: Degraded RNA.
Solution: Your RNA may have been degraded after extraction. Check the integrity of your RNA with an agarose gel run.
Possible Cause: Short reverse transcription time.
Solution: If you wish to amplify long RNA segments, check that your RNA is incubated with your reverse transcriptase for sufficient time.
Possible Cause: Secondary structure on the RNA.
Solution: Increase the cDNA synthesis temperature.
Possible Cause: Wrong priming method.
Solution: 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.
Possible Cause: High dNTP concentration.
Solution: Make sure the final concentration is 0.5 mM or less.
Possible Cause: RNA contains inhibitors of cDNA synthesis.
Solution: Use ethanol or LiCl precipitation to remove these impurities, or do a new extraction round.
Problem: Amplified Product Larger Than Expected
Solution: The sample obtained from RNA extraction might be contaminated with genomic DNA. Make sure to treat your sample with DNase I.
Planning to repeat this experiment?
• Use filter tips for your RNA extraction,
Consider these tips!
• Use RNase decontamination products to clean your work space beforehand,
• Only use RNase-free water for your extraction,
• Start your cDNA synthesis immediately after your RNA extraction,
• Check the quality of your RNA extraction before starting your cDNA synthesis.