Performance in omics analyses of blood samples in long-term storage: opportunities for the exploitation of existing biobanks in environmental health research.
Author(s): Hebels DG, Georgiadis P, Keun HC, Athersuch TJ, Vineis P, Vermeulen R, Portengen L, Bergdahl IA, Hallmans G, Palli D, Bendinelli B, Krogh V, Tumino R, Sacerdote C, Panico S, Kleinjans JC, de Kok TM, Smith MT, Kyrtopoulos SA
Publication: Environ Health Perspect, 2013, Vol. 121, Page 480-7
PubMed ID: 23384616 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of anticoagulant type, delayed centrifugation, temperature of frozen storage, long term frozen storage and RNA stabilization method on the transcriptomic profile and DNA methylation of buffy coat and the metabolomic profile of plasma.
Conclusion of Paper
Anticoagulant type and delayed centrifugation had significant effects on the transcriptome and DNA methylation of buffy coat and plasma metabolomic profile, but the effects on the transcriptome were accompanied by a high false discovery rate (FDR) and effects on the DNA methylation were not systemic. The few effects of frozen storage temperature and duration on the transcriptome of buffy coat specimens were attributable to false positives. RNA yield and RNA integrity numbers (RINs) were higher for RNA from buffy coats thawed with RNAlater than with QIAzol, and RNA yields were greater from specimens anticoagulated with citrate rather then EDTA or heparin, but no other effects of pre-centrifugation storage or anticoagulant type on DNA or RNA quality were observed.
Studies
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant type, delayed centrifugation, temperature of frozen storage, long term frozen storage and RNA stabilization method on RNA yield and integrity as well as transcriptomic profile by Agilent Microarrays. Blood from 4 healthy donors was collected into the three tube types and stored at room temperature for up to 24 h before centrifugation. After centrifugation buffy coats were frozen at -80°C and in liquid nitrogen. For RNA extraction, buffy coats were thawed in RNAlater or QIAzol and extracted using RiboPure blood. Specimens were compared with those from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Italy and North Sweden Health and Disease Studies (NSHDS).
Summary of Findings:
RNA yield and RINs were higher for RNA from buffy coats thawed in RNAlater than in QIAzol (p<0.00001, both), and RNA yields were greater from specimens anticoagulated with citrate rather then EDTA or heparin (p<0.05), but pre-centrifugation storage did not affect RINs or RNA yields, and anticoagulant type had no effect on RINs. In principle component analysis, with the exception of one specimen at one time-point, the first 3 components provided clear separation of the 4 specimens, but there was a trend toward an effect of delayed centrifugation and separation based on anticoagulant type. 3372 of 27181 genes were differentially expressed across the timepoints, >3000 of 28478 among anticoagulants and 2193 of 27552 between the two frozen storage temperatures, but only the difference between specimens stored for 4 versus 8 h before centrifugation and those in EDTA versus heparin were larger than the false discover rate (FDR). Transcripts displaying temporal changes tended to be involved in apoptosis, DNA repair, or stress signaling. Using EPIC-Italy and NSHDS biobanked specimens, only 14 and 76 of the 29662 transcripts, respectively, were significantly affected by storage of 13-17 years (p<0.0033), and those were likely attributable to false positives. Interestingly, a comparison of 6 low and 6 high RIN specimens did not identify significant differences in gene expression.
Biospecimens
Preservative Types
- Frozen
- RNAlater
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA DNA microarray RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Acquisition Anticoagulant Citrate
EDTA
Heparin
Storage Storage duration 0 years
13-17 years
Storage Time at room temperature 0 h
2 h
4 h
8 h
24 h
Storage Storage temperature -80°C
Liquid nitrogen
Analyte Extraction and Purification RNase inactivation QIAzol
RNAlater
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant type, delayed centrifugation, temperature of frozen storage, and long-term frozen storage on DNA yield and integrity from buffy coat specimens as well as DNA methylation profile by Illumina BeadChip. Blood from 4 healthy donors was collected into the three tube types and stored at room temperature for up to 24 h before centrifugation. After centrifugation, buffy coats were frozen at -80°C and in liquid nitrogen. DNA was extracted using the QIAamp Blood mini kit. Specimens were compared with those from the EPIC-Italy and NSHDS.
Summary of Findings:
DNA yield and quality were not affected by anticoagulant type or storage duration, and the effects of delayed centrifugation on the CpG methylation were generally less than those between technical replicates. Nevertheless, 0.6% of interrogated CpG sites showed a >20% variation between time-points (p<0.05), but the effects were reported to be donor-dependent and non-systemic. Importantly, similar CpG profiles were obtained from the biobanked specimens and the fresh specimens, but significant storage time-dependent effects on DNA methylation were only observed for 50 and 1 of 485,764 CpG sites in the EPIC-Italy and NSHDS cohorts.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA DNA microarray DNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Citrate
EDTA
Heparin
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration 0 years
14-17 years
Storage Storage temperature -80°C
Liquid nitrogen
Storage Time at room temperature 0 h
2 h
4 h
8 h
24 h
-
Study Purpose
The purpose of this study was to determine the effects of anticoagulant type, delayed centrifugation, temperature of frozen storage, and long-term frozen storage on the metabolomic profile of plasma by UPLC-ToFMS and by targeted magnetic bead assays. Blood from 4 healthy donors was collected into the three tube types and stored at room temperature for up to 24 h before centrifugation. Specimens were compared with those from the EPIC-Italy and NSHDS.
Summary of Findings:
The metabolomic profile displayed a clear separation between individuals (RSD=18%) and between heparin and citrate or EDTA (RSD=11.7%). Within each anticoagulant group, differences based on delayed centrifugation and donor were observed. Importantly, the number of peaks affected by anticoagulant and centrifugation delay were greater then the FDR. In particular, specimens subjected to an 8 or 24 h delay in centrifugation grouped distinctly from those stored for ≤ 4 h. Targeted proteomic analysis confirmed the deviation of the 8 and 24 h delayed centrifugation specimens from those stored for ≤ 4 h, and an effect of anticoagulant type (citrate or EDTA versus heparin). No clear trend of freezing temperature, or duration of frozen storage on the metabolomic profile of plasma was observed.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Immunoassay Small molecule LC-TOF-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Citrate
EDTA
Heparin
Storage Storage duration 0 years
13-17 years
Storage Storage temperature -80°C
Liquid nitrogen
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Time at room temperature 0 h
2 h
4 h
8 h
24 h