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

The Impact of Tube Type, Centrifugation Conditions, and Hemolysis on Plasma Circulating MicroRNAs.

Author(s): Pastor-Navarro B, Ramírez-Calvo M, Gil-Aldea I, Cortell-Granero I, López-Guerrero JA

Publication: Diagnostics (Basel), 2024, Vol. 14, Page

PubMed ID: 39518337 PubMed Review Paper? No

Purpose of Paper

This paper compared the levels of five cell-free microRNAs (miRNA, miR) and hemolysis in EDTA plasma obtained by double centrifugation and plasma from blood collected in Streck Cell-Free DNA BCT that was centrifuged once or twice.

Conclusion of Paper

Levels of all five miRNAs were comparable in EDTA plasma and Streck plasma centrifuged twice, but  levels of miR-21, miR-182, and miR-125b were higher in Streck plasma centrifuged only once than in EDTA plasma (P<0.0001, P=0.0002 and P=0.03, respectively) or Streck plasma that were centrifuged twice (P<0.0001, P<0.0001 and P=0.0002, respectively). After normalization to miR-16, Streck plasma centrifuged once had higher levels of miR-21 and miR-182b and lower levels of miR-375 than EDTA plasma (P=0.0002, P=0.0002 and P=0.03, respectively) and more miR-182b and less miR-375 than Streck plasma centrifuged twice (P=0.002 and P=0.0002, respectively). Importantly, miRNA levels were not correlated between any two of the three specimen types (EDTA plasma and Streck plasma centrifuged once or twice). While no difference in the absorbance at 414 nm was observed among the specimens during initial analysis, absorbance at 414 nm was higher in EDTA plasma after removal of two Streck specimens due to hemolysis, indicating that EDTA plasma was more susceptible to hemolysis than Streck plasma centrifuged once or twice (P<0.03, both). miR-16 levels were not significantly correlated with the absorbance at 414 nm in any of the specimens examined.

Studies

  1. Study Purpose

    This study compared the levels of five cell-free miRNAs and hemolysis in EDTA plasma that was obtained by double centrifugation and plasma from blood collected in Streck Cell-Free DNA BCT and centrifuged once or twice. Blood was collected from 11 healthy men (39-59 years of age) into Streck Cell-Free DNA BCT tubes and S-Monovette EDTA tubes. EDTA blood was stored for 30 min at 4°C and Streck blood for 4 h at room temperature before plasma separation by centrifugation at 1600 g for 10 min at room temperature. A second centrifugation of the supernatant at 16,000 g for 10 min at 4°C was performed for EDTA plasma and half of each Streck plasma specimen (the remainder was directly frozen).  Plasma was stored at −80°C and thawed at room temperature for 15 min with gentle shaking before RNA isolation with the miRNeasy Serum/Plasma Kit. RNA yield and purity were assessed using a spectrophotometer. Levels of miR-16, miR-21, miR-182, miR-125b, and miR-375 were quantified by real-time RT-PCR with a preamplification step.

    Summary of Findings:

    Levels of all five miRNAs were comparable in EDTA plasma and Streck plasma that were centrifuged twice, but levels of miR-21, miR-182, and miR-125b were higher in Streck plasma that was centrifuged only once than in EDTA plasma (P<0.0001, P=0.0002 and P=0.03, respectively) or Streck plasma that were centrifuged twice (P<0.0001, P<0.0001 and P=0.0002, respectively). Levels of miR-375 were slightly but not significantly lower in Streck plasma centrifuged once than in plasma centrifuged twice (EDTA or Streck). After normalization to miR-16, Streck plasma centrifuged once had higher levels of miR-21 and miR-182b and lower levels of miR-375 than EDTA plasma (P=0.0002, P=0.0002 and P=0.03, respectively) and more miR-182b and less miR-375 than Streck plasma centrifuged twice (P=0.002 and P=0.0002, respectively). Importantly, miRNA levels were not correlated between any two of the three specimen types (EDTA plasma and Streck plasma centrifuged once or twice). While no difference in the absorbance at 414 nm was observed among the specimens during initial analysis, absorbance at 414 nm was higher in EDTA plasma after removal of two Streck specimens due to hemolysis, indicating that EDTA plasma was more susceptible to hemolysis than Streck plasma centrifuged once or twice (P<0.03, both).. miR-16 levels were not significantly correlated with the absorbance at 414 nm.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Protein Spectrophotometry
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
    Biospecimen Acquisition Type of collection container/solution S-Monovette EDTA tube
    Streck Cell-Free DNA BCT

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