Differences in serum and plasma levels of microRNAs and their time-course changes after blood collection.
Author(s): Wakabayashi I, Marumo M, Ekawa K, Daimon T
Publication: Pract Lab Med, 2024, Vol. 39, Page e00376
PubMed ID: 38463196 PubMed Review Paper? No
Purpose of Paper
This paper used microarrays to compare levels of 2,632 miRNAs (miRNA, miR) between serum and plasma and among serum and plasma obtained from blood that was stored at room temperature for up to 180 min before separation.
Conclusion of Paper
Of the 2,632 miRNAs assayed, 985 were detected in both serum and plasma, and 823 were detected at all five timepoints. A total of 299 of the 985 miRNAs detected in both serum and plasma were differentially expressed in the two specimen types at the 0 min timepoint (no additional storage after a 30 min clot for serum). Of those 299 miRNA, the majority (249) were expressed at significantly higher levels in serum than plasma. While levels of erythrocyte-derived miRNAs were generally comparable in serum and plasma, six platelet-derived miRNAs were found at higher levels in plasma, but significance was limited to just three of these. Storage before plasma separation significantly affected levels of 305 miRNAs, 173 of which generally decreased whereas 6 increased with progressive storage. In contrast, delayed separation of serum did not significantly affect the levels of any of the 823 miRNAs analyzed. The authors conclude that clot formation does not alter levels of erythrocyte-derived miRNAs, but serum and plasma miRNA profiles are not interchangeable, and rapid separation of plasma is necessary.
Studies
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Study Purpose
This study used microarrays to compare levels of 2,632 microRNAs (miRNA, miR) between serum and plasma and among serum and plasma obtained from blood that was stored at room temperature for up to 180 min before separation. Blood was collected from five healthy Japanese men (aged 37.2 ± 10.4 years) after an overnight fast; blood was collected into matched Terumo evacuated plastic tubes with a silica-coated film and Terumo K2EDTA tubes. All tubes were stored at room temperature for 0, 15, 30, 60, and 180 min before isolation of serum/plasma; for serum, the storage timecourse began after a 30 min clot time at room temperature. Serum and plasma were separated by centrifugation at 2300 × g for 10 min and at 1200 x g for 10 min, respectively. Serum and plasma were frozen at -80°C until RNA extraction. RNA was extracted using the 3D-Gene RNA Extraction Kit, evaluated using a bioanalyzer, and levels of 2632 miRNAs were quantified by hybridization to 3D-Gene miRNA Oligo Chip. P-values were calculated by Student’s T-test and served as the basis for the estimated q-values. A q-value <0.05 was considered significant.
Summary of Findings:
Of the 2,632 miRNAs assayed, 985 were detected in both serum and plasma, and 823 were detected at all five pre-centrifugation storage timepoints. A total of 299 of the 985 miRNAs detected in both serum and plasma were differentially expressed between the specimen types at the 0 min timepoint (no additional storage after 30 min clot for serum). Of these 299 miRNAs, the majority (249) were expressed at higher levels in serum than plasma. The authors postulate that some of the miRNAs detected at higher levels in serum are likely due to clot formation. All six platelet derived miRNAs examined (miR-185-5p, miR-22-3p and miR-320b) displayed higher levels in plasma than serum; while three of these miRNAs were significantly higher (miR-185-5p, miR-22-3p and miR-320b), the remaining three (let-7b-5p, miR-17-5p and miR-24-3p) displayed a >2-fold differences despite being non- significant (q>005). In contrast, levels of five of the erythrocyte-derived miRNAs were comparable in serum and plasma (miR-25-3p, miR-451a, miR-486-5p and miR-92a-3p), indicating hemolysis did not occur during clot formation; however, one of the erythrocyte-derived miRNAs (miR-423-5p) was found to be expressed at slightly lower levels in plasma than serum (0.72-fold, q=0.0127).
Storage before plasma separation significantly affected levels of 305 miRNAs, of which 173 generally decreased and 6 increased with progressive storage. The authors attribute the decline in levels of miRNA after storage to endocytosis. Levels of three platelet-derived miRNAs (miR-423-5p, miR-92a-3p and miR-22-3p) were among those significantly affected in plasma by the duration of pre-centrifugation, as miR-423-5p and miR-92a-3p decreased with storage. In contrast, delayed separation of serum did not significantly affect the levels of any of the 823 miRNAs analyzed. The authors conclude that clot formation does not alter levels of erythrocyte-derived miRNAs, but serum and plasma miRNA profiled are not interchangeable, and rapid separation of plasma is necessary.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Storage Time at room temperature 15 min (after 30 min clot time for serum)
30 min (after 30 min clot time for serum)
60 min (after 30 min clot time for serum)
180 min (after 30 min clot time for serum)
0 min (after 30 min clot time for serum)
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated