Long-Term Storage Impacts Blood DNA Yield, but Not Integrity or Methylation

Genomic DNA extracted from whole blood is a valuable resource. Ideally, extraction will occur immediately after collection of the blood sample, but this is not always possible for reasons of logistics and economics. This means that whole-blood specimens may undergo prolonged storage—for months or even years—before processing. Inadequate storage, particularly in the case of long-term storage, can negatively impact DNA extraction yield and quality, ultimately resulting in waste or inaccurate experimental results.

For this reason, Bulla et al. (2016) recently investigated the yield and quality of genomic DNA extracted from whole blood after prolonged storage at different temperatures, both with and without a preservative agent.¹ To do this, they collected 41 whole-blood samples from eight healthy volunteers. The research team divided these into three groups: ethylenediaminetetraacetic acid (EDTA) blood, EDTA blood plus preservative at time of storage and EDTA blood plus preservative before thawing, and stored samples from each group at −80°C, −20°C, +4°C and room temperature for one year.

The team first applied UV spectrophotometry with a NanoDrop spectrophotometer (Thermo Scientific) to measure the concentration of the extracted genomic DNA. They reported that EDTA whole-blood samples stored at +4°C and room temperature showed progressive decreases in DNA extraction yield over time, but this process occurred more slowly at the lower temperature. They did not observe this progressive decrease for the EDTA whole-blood samples stored at −20°C and −80°C. At −20°C, they observed relative yields between 50% and 60% over the entire time course, whereas storage at −80°C produced yields of >70%.

The team further reports the preservative agent to be of mixed effectiveness, depending on the storage conditions. EDTA whole-blood samples stored at room temperature with preservative produced relative yields between 60% and 80% over the year. When the researchers added the agent before freezing at −80°C, they observed relative extraction yields of >85% across the time course. However, the same procedure at −20°C produced a decrease in yield over time, ending at ~30% of the baseline after one year of storage. The reason for this difference is unclear. Of particular interest, the research team observed relative yields of >90% at all time points at both −80°C and −20°C when they added the preservative to frozen EDTA blood samples just before thawing.

The researchers also applied spectrofluorometry using a Quant-iT PicoGreen dsDNA assay kit (Thermo Scientific) to measure DNA concentrations and calculate relative yields. Overall, they report results similar to the UV spectrophotometric data. Again, they observed a rapid, significant decrease in DNA extraction yield with samples stored at room temperature. At the other temperatures, they observed decreases at all time points in comparison with the baseline, although relative yields were >60% in all cases.

Their findings regarding samples with preservative were also similar. They report relative yields of >80% for the entire time course at room temperature. Adding the preservative before storage at −80°C produced yields at baseline levels for all time points. However, again samples stored at −20°C with preservative showed decreased yield down to 38% after one year of storage. Also similar to the previously described data, the addition of preservative just before thawing resulted in high yields at all time points for both −80°C and −20°C samples.

Finally, the team analyzed DNA integrity via electrophoresis and polymerase chain reaction assay as well as DNA methylation using a 22-gene panel. They report no significant impact on either measure regardless of storage conditions.

In summary, Bulla et al. indicate that storage temperature significantly influences genomic DNA extraction yield over one year of storage. The addition of a preservative agent to samples stored at room temperature or −80°C positively impacted yield, while the same procedure with samples stored at −20°C negatively impacted yield, for unknown reasons. The authors assert this data is of particular importance to facilities that store whole-blood samples at −20°C. They recommend immediate storage at −80°C, but when this is not feasible, short-term storage at +4°C or at room temperature with a preservative agent can be a suitable alternative.

Reference
1. Bulla, A., et al. (2016) “Blood DNA yield but not integrity or methylation is impacted after long-term storage,” Biopreservation and Biobanking 14(1) (pp. 29–38), doi: 10.1089/bio.2015.0045.