Preventing Degradation in Nucleic Acid Samples
By: Roche Life Science
Posted: January 01, 2018 | Lab Life - DNA & RNA Purification
Although PCR testing can amplify even small quantities of nucleic acid, amplification can fail if the material has degraded due to contamination or degradation. While nuclease contamination is certainly a concern, sample degradation is the more likely threat to success.
Water and oxygen are enemies of nucleic acid, leading to hydrolysis and oxidation. While some degradation over time is inevitable, the goal is to minimize that degradation to ensure that each sample is as “fresh” as possible.
What is the best method for degradation-free storage of nucleic acid in your lab? The answer depends on a variety of factors.
Damage from freezing and thawing
Nucleic acid can be easily damaged during the temperature fluctuations inherent in freeze-thaw cycles. There are three primary means of degradation caused by the temperature change that occurs in the freeze-thaw process:
- Physical shear: As ice crystals form, they can cut through the nucleic acid strands. This can be mitigated by adding glycerol as a cryoprotectant, to reduce the sharp crystals that form in freezing water.
- Oxidative damage: Free radical damage can be caused by iron or other metal ions present in liquid environments. Chelating agents can react with the metal ions to stabilize the solution and minimize the risk to the nucleic acid sample.
- pH changes: Nucleic acids respond best in slightly alkaline conditions, with pH of 7.5-8.0. The freezing and thawing process can change the pH, damaging the sample. TE buffers can help to protect against these pH swings throughout the freeze-thaw cycle for better sample quality.
For samples that need to be used frequently, it’s wise to aliquot larger samples into smaller batches for freezing, reducing the number of times that each batch undergoes the freeze-thaw cycle.
Time and temperature
How long will you be storing your nucleic acid? Each lab has different needs for sample archiving, varying from a few days to many years. In each scenario, labs must preserve samples not just for ideal conditions, but also to protect against temperature fluctuations that could occur during failures of refrigeration or power.
- For immediate use: Short term storage of a few weeks may be accomplished using refrigeration at 4°C. Keeping the sample above freezing eliminates the risks associated with freezing and thawing samples. The use of a TE buffer further protects the sample from degradation.
- Mid-range protection: For mid-term storage — months or years— temperatures between -20°C and -80°C, along with the aid of a TE buffer, may be sufficient to protect the sample for the duration of the research period.
- For sample archiving and storage: Both dry state storage and cryopreservation at -196°C can preserve samples for the long haul — on the order of hundreds of years, if temperatures are maintained. However, if moisture is introduced, or if the temperature increases, the sample could be compromised. Dry state storage would seem to be a reasonable option for protection from the elements, however laboratory plastic tubes and plate seals are not generally airtight. The addition of trehalose, an alpha-linked disaccharide, can help to stabilize these samples and protect against loss by aggregation.
Regardless of the length of storage, using additive buffers can provide an additional level of protection during any temperature change. They can help to minimize degradation and ensure the best possible sample quality for your testing.
The best insurance against degradation
The best protection for your sample is a multi-pronged approach. Aliquot your samples to minimize refreezing; protect the sample with additive buffers; and select the storage temperature that’s appropriate for the length of time that it will be stored. This smart combination will minimize the risk of sample degradation, and ensure that your sample is ready when you are.