Blood DNA Yield but Not Integrity or Methylation Is Impacted After Long-Term Storage.
Author(s): Bulla A, De Witt B, Ammerlaan W, Betsou F, Lescuyer P
Publication: Biopreserv Biobank, 2016, Vol. 14, Page 29-38
PubMed ID: 26812548 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of storage duration of EDTA blood at room temperature, 4˚C, -20˚C, or -80˚C on DNA yield, DNA integrity, and methylation rates. The paper also investigated if adding DNAgard before room temperature or frozen storage or before thawing frozen specimens could attenuate these effects.
Conclusion of Paper
DNA yield declined with storage of blood at room temperature or 4˚C and declined with freezing but the magnitude of the decline was temperature-dependent with smaller changes noted at lower temperatures. Adding DNAgard before storage at room temperature or before thawing frozen specimens stabilized the DNA, but addition of DNAgard prior to storage at -20˚C increased the rate of degradation. As expected, fluorometry showed lower concentrations dsDNA than total DNA. Electrophoresis confirmed modest DNA degradation after storage at room temperature for 7 or 14 days or at -20˚C with DNAgard for one year, but for all other storage durations and temperatures the gel patterns were comparable and long-range PCR possible (15 kb) for all specimens. Variation in the percentage methylation between the baseline specimen and the specimens stored frozen at -20 or -80˚C for 12 months or at room temperature with DNAgard for 12 months was <1%.
Studies
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Study Purpose
This study investigated the effects of storage duration of EDTA blood at room temperature, 4˚C, -20˚C, or -80˚C on DNA yield, DNA integrity, and methylation rates. The study also investigated if adding DNAgard before room temperature or frozen storage or before thawing frozen specimens could attenuate these effects. Blood from eight healthy donors was collected into K2EDTA Vacutainer tubes. Untreated blood was stored at room temperature for 0 h, 24 h, 48 h, 7 days, or 14 days; at 4˚C for 24 h, 48 h, 7 days, 14 days, or 6 months; or at -20˚C or -80˚C for 7 days, 14 days, 6 months, or 12 months before DNA extraction. To test if DNAgard could protect the DNA, DNAgard Blood was added to blood specimens before storage at room temperature for 7 days, 14 days, 6 months, and 12 months and at -20˚C and -80˚C for 7 days, 14 days, 6 months, and 12 months. Additional specimens were stored frozen at -20˚C and -80˚C for 7 days, 14 days, 6 months, and 12 months and DNAgard was added prior to thawing. Blood specimens were thawed at 37˚C and then immediately placed on ice. DNA was extracted using the DNeasy Blood and Tissue Kit with a QIAcube. DNA quantity and quality were determined by spectrophotometry and yield of double-stranded DNA using the Quant-iT PicoGreen dsDNA Assay Kit. The integrity of the DNA was analyzed using gel electrophoresis and by long-range PCR using the GoTaq Long PCR Master Mix Kit. Methylation was evaluated using the Epitect Methyl II Human Stress and Toxicity PCR Arrays in fresh specimens and specimens stored frozen or at room temperature with DNAgard for 12 months. White blood cell counts were performed on a hematology autoanalyzer.
Summary of Findings:
The white blood cell counts were comparable between specimens (5.8 ± 0.6 g/L), but the yield of DNA varied greatly among the specimens even when not subjected to storage (10.9-24.6 µg/mL) with a mean CV of 6%. DNA yield declined with storage of blood at room temperature or 4˚C, but while only 49% of the DNA remained after 7 days at room temperature ,76% remained after 7 days at 4˚C. The shortest time-point examined for frozen storage (7 days) showed lower yields than were obtained in immediately processed specimens (72% at -20˚C and 68% at -80˚C), but levels did not decline much further over 12 months (53% and 73% of baseline after storage at -20 and -80˚C, respectively). Adding DNAgard before storage at room temperature stabilized the DNA such that levels ranged from 63-82% of baseline after 12 months with no clear trend in the loss. Interestingly, while DNAgard provided some protection against freezing effects (79% and 87% of baseline after 1 week at -20 and -80˚C, respectively) and modest stabilization at -80˚C (86% of baseline after 1 year), it did not stabilize DNA during storage at -20˚C (36% of baseline at 1 year). In contrast, adding DNAgard to blood after storage at -20 or -80˚C appeared to provide the best stabilization (92% and 96% of baseline after a year, respectively). As expected, fluorometry showed lower concentrations of double-stranded DNA than total DNA but the overall trends of temperature were similar with DNAgard providing some stabilization when added before or after storage at -80˚C but not when added before storage at -20˚C.
Electrophoresis confirmed some degradation after storage at room temperature for 7 or 14 days and at -20˚C with DNAgard for one year, but for all other storage durations and temperaturesthe gel patterns were comparable for all specimens. Further, long-range PCR allowed for amplification of the 15 kb product in all specimens. Variation in the percentage of methylation between the baseline specimen and the specimens stored frozen at -20 or -80˚C for 12 months or at room temperature with DNAgard for 12 months was <1%.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Bisulfite conversion assay DNA Fluorometry DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature Room temperature
4˚C
-20˚C
-80˚C
Storage Storage duration 0 days
24 h
48 h
7 days
14 days
6 months
12 months
Spectrophotometry Specific Technology platform Fluorometry
Storage Storage conditions With DNAgard
Without DNAgard
Storage Thaw temperature/condition DNAgard added before thaw
No DNAgard added