Related Experiment Video
Updated: Feb 22, 2026
Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification
Published on: November 28, 2016
Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma
Helle Glud Binderup1, Kim Houlind2,3, Jonna Skov Madsen1,3
1Department of Clinical Immunology and Biochemistry, Lillebaelt Hospital, Kolding, Denmark.
Background:
In the past few years, an increasing number of studies have reported the potential use of microRNAs (miRNA) as circulating biomarkers for diagnosis or prognosis of a wide variety of diseases. There is, however, a lack of reproducibility between studies. Due to the high miRNA content in platelets this may partly be explained by residual platelets in the plasma samples used. When collecting fresh plasma samples, it is possible to produce cell-free/platelet-poor plasma by centrifugation. In this study, we systematically investigated whether biobanked EDTA plasma samples could be processed to be suitable for miRNA analysis.
Materials And Methods:
Blood samples were collected from ten healthy volunteers and centrifuged to produce platelet-poor-plasma (PPP) and standard biobank plasma. After one week at -80 °C the biobanked EDTA plasma was re-centrifuged by different steps to remove residual platelets. Using RT-qPCR the levels of 14 miRNAs in the different plasma preparations were compared to that of PPP.
Results:
We were able to remove residual platelets from biobanked EDTA plasma by re-centrifugation of the thawed samples. Nevertheless, for most of the investigated miRNAs, the miRNA level was significantly higher in the re-centrifuged biobanked plasma compared to PPP, even when the platelet count was reduced to 0-1×109/L.
Conclusion:
We found, that pre-storage centrifugation conditions have a significant impact on the measured EDTA plasma level of miRNAs known to be present in platelets. Even for the miRNAs found to be less effected, we showed that a 1.5-3 fold change in plasma levels may possible be caused by or easily overseen due to sample preparation and/or storage.
Insights
Residual platelets in biobanked plasma significantly impact microRNA (miRNA) measurements. Even after re-centrifugation, platelet-derived miRNAs inflate results, affecting biomarker reliability.
Area of Science:
- Biochemistry
- Molecular Biology
- Biomarker Discovery
Background:
- MicroRNAs (miRNAs) show promise as circulating biomarkers for disease diagnosis and prognosis.
- Lack of reproducibility in miRNA studies is a significant challenge.
- Residual platelets in plasma samples contribute to variability in miRNA detection.
Purpose of the Study:
- To investigate the suitability of biobanked EDTA plasma for miRNA analysis.
- To systematically assess the impact of residual platelets on miRNA quantification.
- To determine if re-centrifugation can yield reliable miRNA data from stored plasma.
Main Methods:
- Collected blood from healthy volunteers to create platelet-poor plasma (PPP) and standard biobank plasma.
- Stored biobanked EDTA plasma at -80°C for one week.
- Re-centrifuged thawed biobanked plasma to reduce residual platelet levels.
- Quantified 14 miRNAs using RT-qPCR and compared levels across plasma preparations.
Main Results:
- Re-centrifugation effectively reduced residual platelets in biobanked EDTA plasma.
- Despite reduced platelet counts, miRNA levels in re-centrifuged plasma were significantly higher than in PPP.
- Platelet-derived miRNAs showed substantial increases in re-centrifuged samples.
Conclusions:
- Pre-storage centrifugation significantly influences measured plasma miRNA levels.
- Platelet contamination in biobanked plasma can lead to 1.5-3 fold changes in miRNA levels.
- Sample preparation and storage conditions critically affect miRNA biomarker reliability.
More Related Videos
08:14MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier MSC for Lung Cancer Screening
Published on: October 26, 2017
10:48Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners
Published on: October 25, 2024