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Stability of Circulating Blood-Based MicroRNAs – Pre-Analytic Methodological Considerations

Glinge, Charlotte, Clauss, Sebastian, Boddum, Kim, Jabbari, Reza, Jabbari, Javad, Risgaard, Bjarke, Tomsits, Philipp, Hildebrand, Bianca, Kääb, Stefan, Wakili, Reza, Jespersen, Thomas, Tfelt-Hansen, Jacob·2017·PLOS ONE
DOI 10.1371/journal.pone.0167969PMC PMC5289450
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Fig 1 Different blood tubes for miRNA analysis. miRNA was isolated and levels of miR-1, miR-21 and miR-29b from either; EDTA-plasma, citrate-plasma, lithium-heparin-plasma, or serum fraction, were detected by RT-qPCR. Values were normalized to a spike-in control, cel-miR-39. Normalized average ΔCT values for each are shown, demonstrating amplification from all sources, except lithium-heparin-plasma. In the remaining samples, the levels of miR-21 and miR-29b did not vary significantly between the blood tubes. Li = lithium, ND = not detectable. A) Copenhagen Cohort. B) Munich Cohort.
Fig 1 Different blood tubes for miRNA analysis. miRNA was isolated and levels of miR-1, miR-21 and miR-29b from either; EDTA-plasma, citrate-plasma, lithium-heparin-plasma, or serum fraction, were detected by RT-qPCR. Values were normalized to a spike-in control, cel-miR-39. Normalized average ΔCT values for each are shown, demonstrating amplification from all sources, except lithium-heparin-plasma. In the remaining samples, the levels of miR-21 and miR-29b did not vary significantly between the blood tubes. Li = lithium, ND = not detectable. A) Copenhagen Cohort. B) Munich Cohort.CC BY · PubMed Central
Fig 2 Stability of miRNA in whole blood incubated at room temperature. Whole blood was collected into EDTA containing tubes and incubated for 0, 4, 8, 12, 24 and 72 h at room temperature before processed into plasma. qRT-PCR was performed. Statistically significant differences in miRNA expression are marked with asterisks (**). **p<0.01. A) miR-21. B) miR-29b.
Fig 2 Stability of miRNA in whole blood incubated at room temperature. Whole blood was collected into EDTA containing tubes and incubated for 0, 4, 8, 12, 24 and 72 h at room temperature before processed into plasma. qRT-PCR was performed. Statistically significant differences in miRNA expression are marked with asterisks (**). **p<0.01. A) miR-21. B) miR-29b.CC BY · PubMed Central
Fig 3 Impact of delayed processing on separated blood fractions. Whole blood was collected into EDTA or serum separator containing tubes and incubated at room temperature for 0h, 24h, and 4 days before processed into plasma (A) and serum (B) or processed into plasma (C) and serum (D) immediately and then incubated at room temperature for 0h, 24h, and 4d. Data presented as normalized average ΔCTvalues±SEM. *p<0.05, **p<0.01, ***p<0.001.
Fig 3 Impact of delayed processing on separated blood fractions. Whole blood was collected into EDTA or serum separator containing tubes and incubated at room temperature for 0h, 24h, and 4 days before processed into plasma (A) and serum (B) or processed into plasma (C) and serum (D) immediately and then incubated at room temperature for 0h, 24h, and 4d. Data presented as normalized average ΔCTvalues±SEM. *p<0.05, **p<0.01, ***p<0.001.CC BY · PubMed Central
Fig 4 Impact of physical disturbance. Whole blood was collected into EDTA or serum separator containing tubes and incubated on a shaker at 30 rpm at room temperature for 0h, 1h, and 8h before processed into plasma (A) and serum (B) or processed into plasma (C) and serum (D) immediately and then incubated on a shaker at 30 rpm at room temperature for 0 h, 1 h, and 8 h. Data presented as normalized average ΔCTvalues±SEM. *p<0.05, **p<0.01, One-way ANOVA with Dunnet post test.
Fig 4 Impact of physical disturbance. Whole blood was collected into EDTA or serum separator containing tubes and incubated on a shaker at 30 rpm at room temperature for 0h, 1h, and 8h before processed into plasma (A) and serum (B) or processed into plasma (C) and serum (D) immediately and then incubated on a shaker at 30 rpm at room temperature for 0 h, 1 h, and 8 h. Data presented as normalized average ΔCTvalues±SEM. *p<0.05, **p<0.01, One-way ANOVA with Dunnet post test.CC BY · PubMed Central
Fig 5 Incubation of plasma at -80°C for up to 9 months. There was no significant change in miRNA concentration up to 9 months after freezing for either miR-21 (A) or miR-29b (B) in plasma (p = 0.11 and p = 0.96). However, the concentration of both miRNAs in whole blood increased during storage at -80°C. *p<0.05, **p<0.01.
Fig 5 Incubation of plasma at -80°C for up to 9 months. There was no significant change in miRNA concentration up to 9 months after freezing for either miR-21 (A) or miR-29b (B) in plasma (p = 0.11 and p = 0.96). However, the concentration of both miRNAs in whole blood increased during storage at -80°C. *p<0.05, **p<0.01.CC BY · PubMed Central
Fig 6 MiRNA stability after repetitive freeze-thaw cycles within the separated fractions. Whole blood was collected into EDTA or serum separator containing tubes, immediately processed into plasma (A) and serum (B) and frozen at -80°C. Samples were thawed one or four times before RNA was isolated and RT-qPCR was performed. *p<0.05, **p<0.01, unpaired student t-test.
Fig 6 MiRNA stability after repetitive freeze-thaw cycles within the separated fractions. Whole blood was collected into EDTA or serum separator containing tubes, immediately processed into plasma (A) and serum (B) and frozen at -80°C. Samples were thawed one or four times before RNA was isolated and RT-qPCR was performed. *p<0.05, **p<0.01, unpaired student t-test.CC BY · PubMed Central
Fig 7 MiRNA levels in the 3 blood fractions; plasma, buffy coat and red blood cells (RBC). The two miRNAs, miR-21 and miR-29b, were differently distributed in the fractions; there are significant higher levels of miR-21 in the buffy coat and RBC compared to plasma, and miR-29b plasma levels are significantly lower than in the buffy coat. Statistically significant differences in miRNA expression are marked with asterisks (*).**p<0.01, ***p<0.001.
Fig 7 MiRNA levels in the 3 blood fractions; plasma, buffy coat and red blood cells (RBC). The two miRNAs, miR-21 and miR-29b, were differently distributed in the fractions; there are significant higher levels of miR-21 in the buffy coat and RBC compared to plasma, and miR-29b plasma levels are significantly lower than in the buffy coat. Statistically significant differences in miRNA expression are marked with asterisks (*).**p<0.01, ***p<0.001.CC BY · PubMed Central

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Citations

Cites

Research works printed but not linked: 26

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Metadata

Authors
Glinge, C., Clauss, S., Boddum, K., Jabbari, R., Jabbari, J., Risgaard, B., Tomsits, P., Hildebrand, B., Kääb, S., Wakili, R., Jespersen, T., & Tfelt-Hansen, J.
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2017-02-02
Volume
12
Issue
2
Pages
e0167969
ISSN
1932-6203
DOI
10.1371/journal.pone.0167969
PMC
PMC5289450
Cited by
351
References
5
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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