miRNA in plasma exosome is stable under different storage conditions.
Author(s): Ge Q, Zhou Y, Lu J, Bai Y, Xie X, Lu Z
Publication: Molecules, 2014, Vol. 19, Page 1568-75
PubMed ID: 24473213 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare the stability of microRNA (miRNA, miR) in plasma and exosomes isolated from plasma following refrigerated and frozen storage, and freeze-thaw cycling.
Conclusion of Paper
Exosomes had higher levels of miR-16 and lower levels of miR-451 compared to plasma, but levels of miR-181a and miR-24 were comparable between the two specimen types. RNA concentrations and individual miRNA levels were more stable in exosomes than plasma after storage of plasma at 4°C or -20°C, or freeze-thaw cycling. Generally, the largest effects on miRNA levels was observed following plasma storage at 4°C for 2 weeks, as storage at -20°C or -80°C was associated with small non-significant changes compared to immediately processed controls. While levels of miR-181a, miR-24, and miR-451 in plasma were very sensitive to storage at 4°C, miR-16 was stable.
Studies
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Study Purpose
The purpose of this study was to determine effects of plasma storage at 4°C for 2 weeks; -20°C for 2 weeks, 3 years and 5 years; and -80°C for 2 months on RNA concentrations and levels of miR-16, miR-181a, miR-24, and miR-451 in plasma and exosomes isolated from plasma. Plasma from 8 healthy patients was pooled and aliquoted for storage at each temperature. After storage, exosomes were isolated using ExoQuick exosome precipitation solution. RNA was extracted from plasma and plasma exosomes using the Qiagen miRNeasy Mini kit.
Summary of Findings:
Exosomes had higher levels of miR-16 and lower levels of miR-451 than plasma (p<0.01, and p<0.05, respectively), but levels of miR-181a and miR-24 were comparable in plasma and exosomes. Exosomal RNA concentrations were unaffected by plasma storage for 2 weeks at 4°C, 5 years at -20°C, or 2 months at -80°C, but plasma RNA concentrations were lower when plasma was stored for 5 years at -20°C (p<0.05) or 2 weeks at 4°C (p<0.01). In exosomes, miR-16 levels were unaffected by any of the plasma storage conditions tested, but slight (<2 cycle) variability in cycle threshold (CT) values was noted for exosomal miR-181a, miR-24, and miR-451. In contrast, plasma miR-181a, miR-24, and miR-451 CT values were substantially higher (10-15 cycles) following plasma storage at 4°C for 2 weeks compared to fresh plasma. Slight variability (<3 cycles) in plasma miR-16, miR-24, miR181a and miR-451 CT values were observed among storage temperatures/durations. When plasma and exosome miRNA levels were compared following plasma storage at 4°C for 2 weeks, miR-181a and miR-24 CT values were 10-15 cycles higher in plasma than exosomes, while miR-451 levels were comparable between specimen types.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 4°C
-20°C
-80°C
Storage Storage duration 0 days
2 weeks
2 months
3 years
5 years
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Refrigeration
Real-time qRT-PCR Specific Targeted nucleic acid miR-16
miR-24
miR-181a
miR-451
Biospecimen Aliquots and Components Blood and blood products Plasma
Plasma exosomes
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Study Purpose
The purpose of this study was to investigate effects of plasma freeze-thaw cycling on RNA concentration and levels of miR-16 and miR-451 in plasma and exosomes isolated from plasma. Plasma from 8 healthy patients was pooled and aliquoted for freeze-thaw cycling. After freeze-thaw cycling at -20°C, exosomes were isolated using ExoQuick exosome precipitation solution. RNA was extracted from plasma and exosomes using the Qiagen miRNeasy Mini kit.
Summary of Findings:
RNA concentrations were lower in plasma that was subjected to a single freeze-thaw cycle when compared to frozen never-thawed plasma controls (p<0.01). Plasma subjected to two freeze-thaw cycles displayed lower RNA concentrations in both plasma and exosomes (p<0.01 and p<0.05, respectively). While miR-16 and miR-451 levels in plasma and exosomes declined with each additional plasma freeze-thaw cycle, the magnitudes of decline were larger in plasma than exosomes (12-18 cycles versus <5 cycles).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
Real-time qRT-PCR Specific Targeted nucleic acid miR-16
miR-451
Biospecimen Aliquots and Components Blood and blood products Plasma
Plasma exosomes