Disparate miRNA expression in serum and plasma of patients with acute myocardial infarction: a systematic and paired comparative analysis.
Author(s): Mompeón A, Ortega-Paz L, Vidal-Gómez X, Costa TJ, Pérez-Cremades D, Garcia-Blas S, Brugaletta S, Sanchis J, Sabate M, Novella S, Dantas AP, Hermenegildo C
Publication: Sci Rep, 2020, Vol. 10, Page 5373
PubMed ID: 32214121 PubMed Review Paper? No
Purpose of Paper
This paper compared the level of hemolysis, microRNA (miRNA, miR) yield and fraction, and levels of specific miRNAs in the serum and plasma of patients with non ST-elevation acute myocardial infarction (NSTEMI) and healthy controls.
Conclusion of Paper
The degree of hemolysis of serum and plasma specimens was comparable and well below established thresholds, regardless of the method of measurement (spectrophotometer or ratio of miR-451 to miR-484). The miRNA yield and fraction were comparable in plasma and serum specimens but yield was significantly higher from NSTEMI than control patients, regardless of specimen type used (serum or plasma). Significant differences in levels of U6, RNU44, RNU48, and miR-191 and CVs of miR-186 and miR-192 were identified among the four specimen types (control plasma, control serum, NSTEMI plasma, NSTEMI serum), so the authors used miR-484 for normalization. The miRNA expression pattern was different in plasma and serum with different miRNAs identified as being differentially expressed between control and NSTEMI patients when plasma was used rather than serum. Interestingly, miR21 was higher in the serum and lower in the plasma of NSTEMI patients than controls.
Studies
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Study Purpose
This study compared the level of hemolysis, miRNA yield and fraction, and levels of specific miRNAs in the serum and plasma of patients with NSTEMI and healthy controls. Blood was collected prior to percutaneous coronary intervention into SST and EDTA Vacutainer tubes from 74 NSTEMI patients at one of two sites (39 from Hospital Clínico Universitario de Valencia and 35 from Hospital Clinic i Provincial de Barcelona) and 20 healthy controls. Blood was stored for 30 min at room temperature before isolation of plasma and serum by centrifugation at 1500 x g for 15 min at 4 °C followed by 3000 x g for 5 min at 4 °C and storage at -80°C. RNA was extracted from serum and plasma using the miRCURY RNA Isolation Kit for Biofluids, quantified using a Qubit fluorometer, and analyzed using the Agilent 2100 Bioanalyzer RNA Pico Chip. RNA was reverse-transcribed using the TaqMan miRNA Reverse Transcription Kit and levels of U6, RNU44, RNU48, miR-186, miR-191, miR-192, miR-451, miR-484, miR-1, miR-133a, miR-208b, miR-21, miR-26a, and miR-499 were quantified using TaqMan MicroRNA Assays. The limit of quantification by real-time PCR was defined as 3.2 cycles below that of the blank. The degree of hemolysis was first investigated visually and then specimens with a score of 0 or 1 for visual hemolysis were analyzed using spectrophotometry and real-time PCR amplification of miR-451 and miR-484.
Summary of Findings:
The average degree of hemolysis was comparable in plasma and serum (0.0076 versus 0.0065 for control and 0.0124 versus 0.0066 for NSTEMI) and remained well below the recommendation of <0.03. Hemolysis as measured by the ratio of miR-451 to miR-485 was also comparable between plasma and serum (1.64 versus 1.73 for control and 1.44 versus 1.9 for NSTEMI) and, while the values were slightly different, they were well below the threshold value of 5. Of the 74 NSTEMI patients, 66 had serum and plasma that were not hemolyzed (<0.03% hemolysis and a ratio of miR-451 to miR-484 <5). The miRNA yield was comparable from plasma and serum but was significantly higher in serum and plasma from NSTEMI than control patients (P<0.0001 for serum and P=0.0003 for plasma). The average and standard deviation of the miRNA fraction were comparable in serum and plasma specimens (33.7 ± 9.7% for serum and 32.2± 5.2% for plasma, P=0.55). However, ANOVA identified significant differences between the four groups (control plasma, control serum, NSTEMI plasma, NSTEMI serum) in levels of U6 (P=0.0126), RNU44 (P=0.0086), RNU48 (P=0.0058), and miR-191 (P=0.0083). No significant differences were observed in the levels of miR-186, miR484, and miR-192 but CV values of miR-186 and miR-192 were high and differed among the groups, so the authors used miR-484 for normalization. The miRNA expression patterns were different in plasma and serum. Plasma and serum from patients with NSTEMI had significantly higher expression of miR-1 and miR-208 than plasma and serum from controls, but the variance in expression in NSTEMI patients was higher in plasma than serum. NSTEMI patients also had significantly higher miR-133a levels and significantly lower miR-26 in serum but not plasma and significantly higher miR-499a levels in plasma but not serum. Interestingly, miR-21 was higher in the serum and lower in the plasma of NSTEMI patients than controls. Further, a significant difference in variance among groups was identified for miR-1, miR-208, miR-21, miR-26, and miR-499a.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Cardiovascular Disease
Platform:
Analyte Technology Platform RNA Fluorometry RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Protein Spectrophotometry Protein Macroscopic observation Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Healthy control
NSTEMI
Real-time qRT-PCR Specific Targeted nucleic acid U6
RNU44
RNU48
miR-186
miR-191
miR-192
miR-451
miR-484
miR-1
miR-133a
miR-208b
miR-21
miR-26a
miR-499
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum