The Impact of the Anticoagulant Type in Blood Collection Tubes on Circulating Extracellular Plasma MicroRNA Profiles Revealed by Small RNA Sequencing.
Author(s): Zhelankin AV, Iulmetova LN, Sharova EI
Publication: Int J Mol Sci, 2022, Vol. 23, Page
PubMed ID: 36142259 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare microRNA (miRNA, miR) profiles of case-matched K2EDTA, citrate-theophylline-adenosine-dipyridamole (CTAD), acid citrate dextrose (ACD-B), and citrate plasma from healthy donors.
Conclusion of Paper
K2EDTA plasma had higher hemolysis scores and lower alpha diversity of miRNA species than other plasma types, but total miRNA concentrations were comparable among plasma types. Principal component analysis (PCA) based on the miRNA profile clustered K2EDTA plasma specimens separately from the other plasma types, but one ACD and one citrate specimen clustered with the K2EDTA specimens. A total of 50 miRNA were differentially expressed between the K2EDTA plasma and at least one other specimen type, the majority were found at higher levels in K2EDTA plasma. Many of the miRNAs found at higher levels in K2EDTA plasma by sequencing and real-time PCR were associated with hemolysis or the presence of red blood cells and the miRNA found at lower levels in K2EDTA plasma included platelet-derived miRNA. Further, the ratio of miR-451 (hemolysis-associated) to miR-23a-3p (platelet-derived) was higher in K2EDTA plasma than the other plasma types evaluated, and the ACD and citrate plasma specimen that clustered with K2EDTA specimens in PCA also had a higher ratio of miR-451 to miR-23a-3p. Modest correlations between hemolysis and the ratio of miR-451 to miR-23a-3p by real-time PCR and sequencing were observed ; however the ratios of miR-451 to miR-23-3p by real-time PCR and sequencing were strongly correlated (ρ=0.873, P<0.001). The authors state discrepancies in miRNA levels between sequencing data and qPCR data were observed.
Studies
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Study Purpose
The purpose of this study was to compare miRNA profiles of case-matched K2EDTA, CTAD, ACD-B, and citrate plasma from healthy donors. Blood was collected from ten healthy participants (five male and five female) into sodium citrate, CTAD, ACD-B, and K2EDTA tubes in that order. Platelet-free-plasma (PFP) was obtained by centrifugation at 1000 g for 10 min followed by 2500 g for 20 min. Hemolysis was analyzed in PFP spectrophotometrically based on absorbance at 414 nm. The remaining PFP aliquots were stored at -80°C. PFP was thawed at room temperature for 5 min followed by 37°C for 1-2 min. miRNA was isolated using the NextPrep Magnazol cfRNA Isolation Kit. miRNA was quantified with the Qubit miRNA Assay. miRNA sequencing libraries were prepared using the NEXTFLEX Small RNA-Seq Kit V3 and sequenced using a NextSeq 500 System. Data was analyzed using the QuickMIRSeq pipeline and differential expression between anticoagulants was evaluated using the EdgeR v. 3.32.1 program. miRNA expression changes were validated using TaqMan real-time PCR assays for hsa-miR-223-3p, miR-126-3p, miR-21-5p, miR-150-5p, miR-16-5p, miR-92a-3p, miR-451a 23a-3p, and miR-30e-5p.
Summary of Findings:
Significantly higher hemolysis scores were observed in K2EDTA plasma than ACD (P=0.00013), citrate (P=0.00013) or CTAD (P=0.00015) plasma, but miRNA concentrations were similar among plasma types. A total of 10 specimens were not included in the miRNA read analysis: four specimens failed library preparation (one of each type/anticoagulant) and six with a low percentage (<15%) of miRNA reads (one ACD, one citrate, two CTAD and two K2EDTA plasma specimens). A total of 342 miRNAs were found with >2 counts/million reads in 10 or more specimens of which 317 (92.7%) were found in all four plasma types. There were 13 miRNAs that were not detected in K2EDTA plasma but were detected in the other plasma types evaluated. Alpha diversity was found to be significantly lower in specimens anticoagulated with K2EDTA than the other anticoagulants evaluated (P<0.05). In K2EDTA plasma the top 20 miRNAs represented 90% of miRNA reads, but the top 20 miRNAs made up 77% (CTAD plasma) to 81% (ACD plasma) of reads in the other plasma types investigated. The most abundant miRNAs in all specimens were miR-451a and miR-486-5p which accounted for 65% of reads in K2EDTA plasma and 35-40% of reads in the other plasma types. A total of 2990 miRNA isoforms of 302 miRNAs were found at >10 counts in >10 specimens, of which 32% matched the canonical sequence and >50% had modifications on the 3’ end (mostly trimmed). PCA based on the miRNA profile clustered the K2EDTA plasma specimens separately from the other plasma types evaluated, but one ACD and one citrate specimen clustered with the K2EDTA specimens. A total of 50 miRNAs were differentially expressed between plasma types, all of which were differently expressed between the K2EDTA plasma and at least one other specimen type. Among the 31 miRNAs found at higher levels in K2EDTA plasma, many were associated with hemolysis or the presence of red blood cells including miR-16-5p, miR-486-5p, miR-484, miR-451a, miR-92a-3p, miR-16-2-3p and miR-25-3p. Analysis of miRNA based on known expression revealed that RBC-derived and hemolysis-associated miRNAs were elevated in K2EDTA plasma and platelet-derived miRNA were found at lower levels. Real-time PCR confirmed a higher ratio of RBC-derived to platelet-derived miRNA in K2EDTA plasma than all other plasma types (P=0.00037, all) and a higher relative level of each of the RBC-derived miRNA and lower levels of platelet-derived miRNA in K2EDTA specimens compared to the other plasma types evaluated (P<0.05). Further, the ratio of miR-451 (hemolysis-associated) to miR-23a-3p (platelet-derived) was higher in K2EDTA plasma than the other plasma types (P<0.001, all). Importantly, the ACD and citrate plasma specimen that clustered with the K2EDTA specimens in PCA also had a higher ratio of miR-451 to miR-23a-3p. Modest correlations between hemolysis and the ratio of miR-451 to miR-23a-3p were observed when determined by real-time PCR (ρ=0.417, P=0.013) and sequencing (ρ=0.513, P=0.004); correlations were strong when the ratio of miR-451 to miR-23a-3p determined by real-time PCR or sequencing were compared (ρ=0.873, P<0.001). The authors state discrepancies in miRNA levels between sequencing data and qPCR data were observed.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Protein Spectrophotometry RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Acid-citrate-dextrose
Citrate-theophylline-adenosine-dipyridamole
Citrate
Potassium EDTA
Next generation sequencing Specific Technology platform Real-time PCR
Spectrophotometry