Optimized Collection Protocol for Plasma MicroRNA Measurement in Patients with Cardiovascular Disease.
Author(s): Wu CS, Lin FC, Chen SJ, Chen YL, Chung WJ, Cheng CI
Publication: Biomed Res Int, 2016, Vol. 2016, Page 2901938
PubMed ID: 27725938 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of delayed centrifugation, storing plasma at room temperature or 4°C, and heparinase treatment on microRNA (miRNA, miR) levels in plasma from patients undergoing cardiac catheterization.
Conclusion of Paper
While miR-451a and miR-23a levels were unaffected by delayed centrifugation, the ratio of mIR-451a to miR-23a increased with increasing hemolysis and had 100% sensitivity and specificity for detection of significant hemolysis. The average of the ΔCT values for 18 miRNAs increased with storage of plasma at room temperature and 4°C but changes observed were larger when storage was at 4°C rather than room temperature for 30 min up to 2 h. Significant reductions in the median CT were observed for miR-15b-5p and miR-30e-5p when plasma was stored at room temperature for 2 h but the other 16 miRNAs were unaffected.
While heparin-plasma had significantly lower quantified levels of cel-miR-39 than plasma without heparin, treatment with 0.25 or 0.5 units of heparinase resulted in levels that were comparable to plasma without heparin. The effects of heparinase on the levels of miR-15b-5p and exogenous cel-miR-39 were dose-dependent, with increasing levels observed with increasing dose (0.025-0.25 units), regardless of storage temperature. Importantly, when heparinized plasma was treated with 0.25 units heparinase, expression levels of 18 miRNAs were unaffected by storage at room temperature or 4°C for 2 or 4 h.
Studies
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Study Purpose
This study investigated the effects of delayed centrifugation, storing plasma at room temperature or 4·°C, and heparinase treatment on microRNA (miRNA, miR) levels in plasma. Heparinized and unheparinized blood were obtained from patients undergoing cardiac catheterization for ischemic heart disease or heart failure. Blood (heparinized and unheparinized) was placed in K2EDTA tubes and stored at room temperature or 4°C for 0.5, 1, 2, 4, or 8 h before centrifugation at 2000 x g for 10 min. The resultant plasma was transferred to a new tube and centrifuged for 2500 x g for 15 min, placed in a new tube containing protease inhibitors, and frozen at -80°C. Hemolyzed specimens were created by shaking 1 mL unheparinized EDTA blood from four patients until the desired level of hemolysis occurred, after which plasma was obtained as described above. miRNA was extracted using the miRNeasy mini kit and quantified using TaqMan real-time PCR assay which the authors validated. To investigate if heparinase treatment would allow for the use of heparinized specimens in real-time PCR assays, 0.0025-0.5 units of heparinase were added to the reverse transcribed RNA. The effects of plasma storage temperature were investigated using plasma from five patients and the effect of 2 h of plasma storage on specific miRNA was investigated using nine patients.
Summary of Findings:
While miR-451a and miR-23a levels were unaffected by storage of blood at room temperature or 4°C for 8 h before centrifugation, the ratio of mIR-451a to miR-23a increased with increasing degree of hemolysis. A threshold ratio for miR-451a to miR-23a of 60 had 100% sensitivity and specificity for detection of significant hemolysis (0.12% or greater). The average of the ΔCT values for 18 miRNAs significantly increased with storage of five plasma specimens at room temperature and 4°C (P<0.0001 and P=0.0368, respectively). The changes in average ΔCT were larger when plasma was stored at 4°C rather than room temperature and although the differences were significant when storage was for 30 min to 2 h (P<0.001, all), they were no longer significant when stored for 4 h (P=0.301). Consequently, the authors recommend plasma be stored at room temperature. Average levels of miRNA in each of the nine patients were not significantly affected by storage of plasma at room temperature for 2 h, but significant reductions in the median CT were observed for miR-15b-5p (P=0.0315) and miR-30e-5p (P=0.0315).
While heparin-plasma had significantly lower quantified levels of cel-miR-39 than plasma without heparin (difference of 1.5-2.8 Cts, P<0.05), treatment with 0.25 or 0.5 units of heparinase resulted in levels that were comparable to plasma without heparin. Treatment of heparin-plasma with 0.25 or 0.5 units of heparinase resulted in comparable levels of miR-15b-5p, miR-17-5p, and miR18e-5p. While the effects of heparinase on the levels of miR-15b-5p and exogenous cel-miR-39 were dose-dependent, with increasing levels observed with increasing dose (0.025-0.25 units), regardless of storage temperature, the significance for miR-15b-5p depended on storage temperature and duration. Importantly, when heparinized plasma was treated with 0.25 units heparinase, expression levels of 18 miRNAs were unaffected by storage at room temperature or 4°C for 2 or 4 h.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Cardiovascular Disease
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Real-time qRT-PCR Specific Template modification 0.0025 units heparinase
0.025 units heparinase
0.25 units heparinase
0.5 units heparinase
No heparinase added
Storage Storage temperature Room temperature
4°C
Storage Storage duration 0 h
0.5 h
1 h
2 h
4 h
8 h