Red blood cells as potential materials for microRNA biomarker study: overcoming heparin-related challenges.
Author(s): Kontidou E, Humoud R, Chernogubova E, Alvarsson M, Maegdefessel L, Collado A, Pernow J, Zhou Z
Publication: Am J Physiol Heart Circ Physiol, 2024, Vol. , Page
PubMed ID: 39422364 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of four microRNAs (miRNA, miR) among EDTA and heparinized red blood cells (RBCs) and plasma. The authors also investigated whether differences in miR-210-3p expression between RBCs from healthy volunteers and patients with type II diabetes were observed with either anticoagulant.
Conclusion of Paper
Levels of miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were comparable in case-matched heparinized and EDTA RBCs. Levels of miR-210-3p were lower in RBCs from patients with type II diabetes relative to healthy volunteers both when the RBC came from EDTA (P<0.01) or heparinized (P<0.05) blood. miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were detected in EDTA plasma, but were not detected in heparinized plasma, which the authors attribute to the known interference of heparin with the assay. EDTA RBCs had much higher levels of miR-451a and miR-16-5p (P<0.001, both) and lower levels of miR-21-5p (P<0.05) than EDTA plasma, but levels of miR-210-3p were comparable between EDTA RBCs and plasma. The authors conclude that RBCs may be a viable option for the analysis of miRNA in heparinized blood.
Studies
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Study Purpose
This study compared levels of four microRNAs (miRNA, miR) among EDTA and heparinized red blood cells (RBCs) and plasma. The authors also investigated whether differences in miR-210-3p expression between RBCs from healthy volunteers and patients with type II diabetes were observed with either anticoagulant. Blood was collected from 13 patients with type II diabetes and 28 healthy volunteers after >12 h fasting; blood was collected into EDTA tubes or sodium heparin tubes. Plasma was separated by centrifugation at 1,000 g for 10 min at 4°C and RNA was isolated using the miRNeasy Serum/Plasma Kit. RBCs were obtained by three consecutive washes in Krebs-Henseleit buffer with centrifugation at 1,000 g for 5 min at 4°C, and RNA was isolated using the miRNeasy Mini Kit. RNA was quantified by spectrophotometry and levels of miR-210-3p, miR-21-5p, miR-16-5p, miR-451a, and U6 were measured by real-time RT-PCR. Sequencing libraries were constructed using the NEB Small RNA Sample Prep Kit and single-end read using an Illumina NovoSeq 6000 platform.
Summary of Findings:
On average, 738 miRNAs were identified in RBCs from EDTA tubes, including miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a. Levels of miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were comparable in case-matched heparinized and EDTA RBCs. Levels of miR-210-3p were lower in RBCs from patients with type II diabetes relative to healthy volunteers both when the RBCs came from EDTA (P<0.01) or heparinized (P<0.05) blood. miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were detected in EDTA plasma, but were not detected in heparinized plasma, which the authors attribute to the known interference of heparin with the assay. EDTA RBCs had much higher levels of miR-451a and miR-16-5p (P<0.001, both) and lower levels of miR-21-5p (P<0.05) than EDTA plasma, but levels of miR-210-3p were comparable between EDTA RBCs and plasma. The authors conclude that RBCs may be a viable option for the analysis of miRNA in heparinized blood.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
- Diabetes Type 2
Platform:
Analyte Technology Platform RNA Next generation sequencing RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant EDTA
Sodium heparin
Real-time qRT-PCR Specific Targeted nucleic acid miR-210-3p
miR-21-5p
miR-16-5p
miR-451a
U6
Biospecimen Aliquots and Components Blood and blood products Plasma
Red blood cells
Preaquisition Diagnosis/ patient condition Type II diabetes
Healthy