microRNA biomarker discovery and high-throughput DNA sequencing are possible using long-term archived serum samples.
Author(s): Rounge TB, Lauritzen M, Langseth H, Enerly E, Lyle R, Gislefoss RE
Publication: Cancer Epidemiol Biomarkers Prev, 2015, Vol. 24, Page 1381-7
PubMed ID: 26108462 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of storage duration, freezing, lyophilization, the additive iodacetate, and clotting time on miRNA and DNA yields and the percentage of miRNA (10-40 nt) of total small RNA (0-150 nt) in serum.
Conclusion of Paper
The average miRNA yield was higher from lyophilized serum than from serum stored at -20°C. Increasing frozen storage may have decreased the percentage of miRNA and increased DNA yield in the health examinations (HE) cohort, but the effects were complicated by the use of the additive iodacetate in the oldest specimens. Importantly, similar effects were not reproduced in a separate specimen cohort of Red Cross blood donors (RCBD) in which the oldest specimens did not contain the additive. The additive iodacetate may have attenuated the effects of frozen storage on DNA yields and miRNA percentages, but did not improve miRNA yields. Differences in DNA yield and miRNA percentage were present between the two specimen cohorts that were attributed to differences in clot time (1 h versus 14-28 h), but differences in storage duration and use of the tube additive iodacetate were confounding variables.
Studies
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Study Purpose
The purpose of this study was to compare miRNA yield from fresh serum, frozen lyophilized serum, and serum frozen with or without the additive iodacetate. Serum specimens were subjected to different storage durations and clotting times. Serum specimens that were collected during a health examination (HE) included 44 specimens collected with the additive iodoacetate (1972-1978), 44 specimens collected with no additives (1979-1986), and 49 specimens collected in gel tubes (1987-2004). HE specimens were centrifuged within 1 h, shipped cold and frozen at -20°C. Serum specimens collected from Red Cross blood donors (RCBD) included 4 specimens collected without additive between 1973-1979, 42 lyophilized specimens collected between 1973-1979, 48 specimens collected without additive between 1980-1990 and 45 specimens collected without additive between 1997-2004. All RCBD specimens were stored overnight (14-28 h clotting time), processed to serum and frozen at -20°C or lyophilized. HE and RBCD specimens had been thawed 1-3 times previously, but potential effects were not explored. Additionally, 43 fresh serum specimens isolated following a 1 h clotting time were included. RNA was extracted using TRIzol LS with the miRNeasy Serum/Plasma Kit. Storage durations were calculated based on specimens collected in 1972 having been stored for 40 years.
Summary of Findings:
Average miRNA yields were higher from lyophilized serum than serum stored at -20°C (p≤0.05), and from fresh serum than frozen serum collected with the additive iodacetate stored for 34-40 years (p=0.023). The percentage of miRNA (10-40 nt) in relation to amount of total small RNA (0-150 nt) was greater in HE specimens collected with the additive iodacetate and stored 34-40 years than in HE specimens that did not contain the additive and were stored 8-25 years (p=0.042) or 26-33 years (p<0.001), indicating a potential effect of storage duration or a protective effect of the additive iodacetate. However, as RCBD specimens, which had similar storage durations (8-15, 22-32 and 33-39 years) but did not contain the additive and had longer clotting time compared to HE specimens (14-28 h versus 1 h) displayed no differences in the percentage of miRNA in relation to small RNA, the effect is more likely attributable to the additive.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Tube containing iodacetate
Gel tube
Plain tube
Storage Storage duration 1 h
14-28 h
0 years
8-15 years
16-33 years
34-40 years
8-25 years
26-32 years
33-39 years
Biospecimen Preservation Type of fixation/preservation Lyophilized
Frozen
None (fresh)
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
The purpose of this study was to compare DNA yield from fresh serum, frozen lyophilized serum and serum frozen with or without iodacetate and to investigate the effects of storage duration and clotting time. Specimens collected during a health examination (HE) included 25 that were collected with the additive iodoacetate (1972-1978), 25 collected with no additives (1979-1986), and 25 collected in gel tubes (1987-2004). HE specimens were centrifuged within 1 h, shipped cold and frozen at -20°C. Specimens collected from Red Cross blood donors (RCBDs) consisted of 4 collected without an additive between 1973-1979, 24 that were collected and lyophilized between 1973-1979, 25 that were collected without an additive between 1980-1990, and 25 that were collected without an additive between 1997-2004. All RCBD specimens were stored overnight (14-28 h clotting time), processed to serum and frozen at -20°C or lyophilized. HE and RBCD specimens had been thawed 1-3 times previously, but potential effects were not investigated. Additionally, 24 fresh specimens with a 1 h clotting time were included. DNA was extracted using the QIAamp blood mini kit. Storage durations were calculated based on specimens collected in 1972 having been stored for 40 years.
Summary of Findings:
DNA yield was lower from fresh specimens than HE specimens stored for 26-33 years (p<0.001) or 8-25 years (p<0.001), as well as RCBD specimens subjected to a longer clotting time (14-28 h versus 1 h) that had been frozen for 8-15 years (p=0.002). DNA yields were comparable between fresh specimens and HE specimens with iodacetate frozen for 34-40 years. Importantly, in frozen HE specimens with a 1 h clot time, DNA yield was higher in serum collected without iodacetate than with iodacetate (p=0.008). DNA yield was also higher in HE specimens collected without iodacetate than frozen RCBD specimens (1 versus 14-28 h clot time, p=0.009) or fresh serum (p<0.001). Frozen HE specimens with the additive iodacetate had comparable DNA yields to fresh specimens collected without the additive(both 1 h clot time). DNA yields were also higher from frozen RCBD specimens than from fresh serum (14-28 h clot versus 1 h clot, p=0.021). The authors report the presence of apoptotic DNA fragmentation on the electropherogram of most specimens. 12 specimens yielded DNA of sufficient quality for next generation sequencing, but the authors do not report which specimens.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA Next generation sequencing DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Tube containing iodacetate
Gel tube
Plain tube
Storage Storage duration 1 h
14-28 h
0 years
8-15 years
16-33 years
34-40 years
8-25 years
26-32 years
33-39 years
Biospecimen Preservation Type of fixation/preservation Lyophilized
Frozen
None (fresh)
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated