NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Storage Stability of Blood Samples for miRNAs in Glycosylated Extracellular Vesicles.

Author(s): Ma C, Ding R, Hao K, Du W, Xu L, Gao Q, Yu C

Publication: Molecules, 2023, Vol. 29, Page

PubMed ID: 38202686 PubMed Review Paper? No

Purpose of Paper

This paper compared the levels of four microRNAs (miRNAs, miR) in glycosylated extracellular vesicles (EVs) that were obtained from serum that was separated using different centrifugation methods and analyzed immediately or after 8 h; from serum that was separated from blood stored at 4°C for up to 48 h or at 25°C for up to 12 h; from serum that was stored at -20°C for up to 90 days, 4°C for up to 6 days, 25°C for up to 24 h, or 37°C for up to 12 h; from fresh serum and serum that was freeze-thaw cycled once; from serum preserved with ribonucleoside–vanadyl complex (RVC) with or without 1% PC300 or with saline; and from plasma that was separated from blood collected in different tube types and stored at 25°C for up to 120 h.

Conclusion of Paper

The particles isolated from serum had a size distribution of 153.1 ± 67.1 nm, had a double layer membrane and cup-shape, and expressed the EV markers CD63, CD81, and TSG101, indicating the EV isolation was successful. Serum miR-16, let-7a, miR-125a, and miR-150 levels were comparable in EVs isolated by two-step centrifugation, a single-step rapid acceleration centrifugation, or a single-step slow acceleration centrifugation when samples were analyzed immediately.  When serum was left in the original tube for 8 h after centrifugation, levels of all four miRNAs analyzed declined and miR-16 levels in serum obtained by a two-step centrifugation were lower than in serum obtained by a single slow acceleration step.

When blood was stored at 4°C for ≥4 h before serum separation, hemolysis was observed. Significant effects on miRNA levels were observed when blood was stored at 4°C for ≥8 h before serum separation, at the first timepoint when serum was stored at 4°C, 25°C or 37°C (1 day, 4 h and 2 h, respectively), and when serum was freeze-thaw cycled once rather than analyzed fresh; however, miRNA levels were not significantly affected by storage of blood at 25°C for 12 h or storage of serum at -20°C for 90 days. Ribonucleoside–vanadyl complex (RVC) (200 mM), with or without PC300, stabilized levels of all four miRNAs in serum that was stored at 25°C for up to 12 h but, as expected, levels of all four miRNAs declined with storage at 25°C when saline was added to serum.  For plasma, significant differences in miRNA levels were noted after storage of blood in EDTA tubes for 48 h or in sodium citrate tubes for 72 h. miRNA levels in plasma were unaffected by storage of blood in Lakebio’s cfRNA preservation tubes for up to 120 h.  

Studies

  1. Study Purpose

    This study compared the levels of four miRNAs in glycosylated EVs that were isolated from serum obtained by three different centrifugation methods and then or frozen either immediately or after storage for 8 h in the original tube. Blood was collected from 10 healthy volunteers into serum tubes and stored for 30-90 min (temperature not specified) before processing. Serum was separated by three different methods: 1) Centrifugation at 1800 g for 10 min at room temperature followed by recentrifugation of serum at 3000 g for 10 min at room temperature immediately or after 8 h at 25°C (two-step centrifugation), 2) Centrifugation of serum at 3000 g with rapid acceleration (mode 9) for 10 min at room temperature and removal of an aliquot of the resultant serum immediately and after 8 h at 25°C (single-step rapid acceleration), and 3). Centrifugation of serum at 3000 g with slow centrifugation for 10 min at room temperature and removal of an aliquot of the resultant serum immediately and after 8 h at 25°C (single-step slow acceleration). All serum aliquots were frozen at -80°C until analysis. Glycosylated EVs were isolated using the Glyexo-Capture method and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western Blot analysis of calnexin, CD63, CD81, and TSG101. RNA was isolated using the miRNeasy Mini Kit and quantified using the Qubit microRNA Assay. Levels of let-7a, miR-16, miR-125a, and miR-150 were quantified by real-time RT-PCR.

    Summary of Findings:

    The particles isolated from serum had a size distribution of 153.1 ± 67.1 nm, had a double layer membrane and cup-shape, and expressed the EV markers CD63, CD81, and TSG101, indicating that EV isolation was successful. Serum miR-16, let-7a, miR-125a, and miR-150 levels were comparable in EVs isolated by two-step centrifugation, a single-step rapid acceleration centrifugation, or a single-step slow acceleration centrifugation. When serum was left in the original tube for 8 h after centrifugation, levels of all four miRNA analyzed declined (36-77% in two-step centrifugation serum, 5-58% in single-step rapid acceleration centrifugation serum, and 13-70% in single-step slow acceleration serum). After post-centrifugation storage for 8 h of, miR-16 levels in serum obtained by a two-step centrifugation were lower than levels in serum obtained by a single slow acceleration step (-36% versus 5%, P<0.05); however, levels of let-7a, miR-125a, and miR-150 did not differ significantly among the three centrifugation methods evaluated.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    RNA Fluorometry
    Protein Western blot
    Morphology Electron microscopy
    Cell count/volume Light scattering
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 0 h
    8 h
    Biospecimen Aliquots and Components Centrifugation Different acceleration rates compared
    Centrifugation delays investigated
    Different number of centrifugation steps compared
  2. Study Purpose

    This study compared the levels of four miRNAs in glycosylated EVs that were obtained from serum separated from blood that was stored at 4°C for up to 48 h or 25°C for up to 12 h; in serum stored at -20°C for up to 90 days, 4°C for up to 6 days, 25°C for up to 24 h, or 37°C for up to 12 h; in fresh serum and serum that was freeze-thaw cycled once; in RVC-preserved serum with or without 1% PC300 or saline; and in plasma from blood that was collected in different tube types and stored at 25°C for up to 120 h. Unless otherwise specified, blood from healthy volunteers was stored for 30-90 min (temperature not specified) before serum or plasma separation by centrifugation at 1800 g for 10 min at room temperature followed by recentrifugation of serum at 3000 g for 10 min at room temperature. Isolated serum or plasma was then frozen at -80°C until analysis. To investigate the effects of delayed processing, blood from ten volunteers was stored at 4°C for 0, 4, 8, 12, 24, and 48 h, or 25°C for 0, 4, 6, 8, 10, and 12 h before serum separation.  To investigate the effects of serum storage, serum from the blood of 12 healthy volunteers was stored at 25°C for 0, 4, 8, 12, 24, and 48 h; at 37°C for 0, 2, 4, 6, 8, 10, and 12 h; 4°C for 0, 1, 2, 3, 4, 5, and 6 days; or −20°C for 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 days before freezing at -80°C. To investigate the effects of serum freeze-thaw cycling, serum from 3 healthy volunteers were aliquoted and one aliquot was used directly for EV isolation (fresh) while the other serum aliquot was frozen at -80°C for 24 h before thawing and EV isolation. To investigate the effects of protectants, matched aliquots of serum aliquots from three volunteers were preserved with 200 mM RVC, 200 mM RVC supplemented with 1% PC300, or 10 × normal saline and stored at 25°C for 0, 6, or 12 h before freezing. To investigate the effects of plasma collection tube type, blood was collected from an unspecified number of healthy volunteers into EDTA, sodium citrate, and Lakebio cell-free RNA storage tubes and stored at 25°C for 0, 4, 8, 16, 24, 48, 72, and 120 h before plasma separation. Glycosylated EVs were isolated using the Glyexo-Capture method. RNA was isolated from EVs using the miRNeasy Mini Kit and quantified using the Qubit microRNA Assay. Levels of let-7a, miR-16, miR-125a, and miR-150 were quantified by real-time RT-PCR.

    Summary of Findings:

    When blood was stored at 4°C for ≥4 h before serum separation, hemolysis was observed. Levels of let-7a were significantly higher when blood was stored at 4°C for ≥8 h before serum separation (P<0.001, all), and non-significant increases in miR-16 and miR-150 and decreases in miR-125a were observed. In contrast, levels of the investigated miRNAs were unaffected by storage of blood at 25°C for up to 12 h before serum separation. Nevertheless, the authors state it is crucial to separate serum from blood within 2 h of collection regardless of the temperature of the delay to centrifugation.

    Levels of the miRNAs of interest declined progressively when  separated serum was stored at 4°C, 25°C, or 37°C, with faster declines noted at 37°C than 25°C and at 25°C than 4°C; nevertheless, at either temperature, a significant decline in the level of at least one miRNA was observed at the earliest timepoint investigated (1 days, 4h and 2h, respectively) relative to immediately analyzed serum. The magnitude of the decrease was miRNA-dependent, and the authors state larger declines were observed in miRNAs with low abundance than miRNAs with higher abundance.  Importantly, miRNA levels were unaffected when separated serum was stored at -20°C for 90 days. Compared to fresh serum, serum that was freeze-thaw cycled once had significantly lower levels (-48 to -70%) of all four miRNA investigated (P<0.05, all). When 200 mM ribonucleoside–vanadyl complex (RVC) with or without PC300 was added to serum, the levels of all four miRNAs stabilized in serum specimens that were stored at 25°C for up to 12 h; but, as expected, levels of all four miRNAs declined with storage at 25°C when saline was added to serum.  For plasma, significant differences in miRNA levels were noted after blood was stored in EDTA tubes for 48 h (miR-125a and miR-150a; P<0.0001, both) or in sodium citrate tubes for 72 h (let-7a and miR-16; P<0.01, both), but miRNA levels in plasma were stable in blood stored in Lakebio’s cfRNA preservation tubes for up to 120 h.  

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration Blood at 4°C for 0, 4, 8, 12, 24, and 48 h
    Serum at 25°C for 0, 4, 8, 12, 24, and 48 h
    Blood at 25°C 0, 4, 6, 8, 10, and 12 h
    Serum at 37°C for 0, 2, 4, 6, 8, 10, and 12 h
    Serum at 4°C for 0, 1, 2, 3, 4, 5, and 6 days
    Serum at −20°C for 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 days
    Plasma at 25°C for 0, 4, 8, 16, 24, 48, 72, and 120 h
    Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
    Biospecimen Acquisition Type of collection container/solution EDTA tube
    Sodium citrate tube
    Lakebio cell-free RNA storage tubes
    Biospecimen Acquisition Anticoagulant EDTA
    Sodium citrate
    Storage Storage temperature Blood at 4°C for 0, 4, 8, 12, 24, and 48 h
    Blood at 25°C 0, 4, 6, 8, 10, and 12 h
    Serum at 25°C for 0, 4, 8, 12, 24, and 48 h
    Serum at 37°C for 0, 2, 4, 6, 8, 10, and 12 h
    Serum at 4°C for 0, 1, 2, 3, 4, 5, and 6 days
    Serum at −20°C for 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 days
    Storage Freeze/thaw cycling 0 cycles
    1 cycle
    Biospecimen Preservation RNA stabilization method Ribonucleoside–vanadyl complex
    None
    PC300

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