Dataset Information

Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

ABSTRACT: Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating microRNA levels are affected by artificial platelet activation due to suboptimal plasma preparation. To address this issue, we used a standardized RT-qPCR test for 12 microRNAs (thrombomiR®, TAmiRNA GmbH, Vienna, Austria) that have been associated with cardiovascular and thrombotic diseases and were detected in platelets and/other hematopoietic cells. Blood was prevented from coagulating with citrate-theophylline-adenosine-dipyridamole (CTAD), sodium citrate, or ethylenediaminetetraacetic acid (EDTA) and stored for different time periods either at room temperature or at 4 °C prior to plasma preparation and the subsequent quantification of microRNAs. We found that five microRNAs (miR-191-5p, miR-320a, miR-21-5p, miR-23a-3p, and miR-451a) were significantly increased in the EDTA plasma. Moreover, we observed a time-dependent increase in plasma microRNAs that was most pronounced in the EDTA blood stored at room temperature for 24 h. Furthermore, significant correlations between microRNA levels and plasma concentrations of platelet-stored molecules pointed towards in vitro platelet activation. Therefore, we strongly recommend to (i) use CTAD as an anticoagulant, (ii) process blood samples as quickly as possible, and (iii) store blood samples at 4 °C whenever immediate plasma preparation is not feasible to generate reliable data on blood-derived microRNA signatures.

SUBMITTER: Mussbacher M

PROVIDER: S-EPMC7464075 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

Mussbacher Marion M Krammer Teresa L TL Heber Stefan S Schrottmaier Waltraud C WC Zeibig Stephan S Holthoff Hans-Peter HP Pereyra David D Starlinger Patrick P Hackl Matthias M Assinger Alice A

Cells 20200818 8

Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating mic ...[more]

PMID: 32824700

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Project description:Pasteurization of donated human milk preserves it for storage and makes it safe for feeding, but at the expense of its composition, nutritional values and functions. Here, we aimed to investigate the impact of Holder Pasteurization (HoP) and High Pressure Processing (HPP) methods on miRNA in human milk and to evaluate impact of these changes on miRNA functions. Milk samples obtained from women in 50th day of lactation (n = 3) were subjected either to HoP, HPP or remained unpasteurized as a control. Subsequently, miRNA was isolated from whole material and exosomal fraction and sequenced with Illumina NextSeq 500. Sequencing data were processed, read counts were mapped to miRNA and analyzed both quantitatively with DESeq2 and functionally with DIANA mirPath v.3. While HPP caused statistically insignificant decrease in number of miRNA reads compared to unprocessed material, HoP led to 82-fold decrease in whole material (p = 0.0288) and 302-fold decrease in exosomes (p = 0.0021) not leaving enough reads for further analysis. Changes in composition of miRNA fraction before and after HPP indicated uneven stability of individual miRNAs under high pressure conditions, with miR-30d-5p identified as relatively stable and miR-29 family as sensitive to HPP. Interestingly, about 2/3 of unprocessed milk miRNA content consists of only 10 distinct miRNAs with miR-148a-3p at the top. Functional analysis of most abundant human milk miRNAs showed their involvement in signaling pathways, cell communication, proliferation and metabolism that are obviously important in rapidly growing infants. Functions of miRNAs which suffered the greatest depletion during HPP were similar to roles of the majority of unprocessed human milk's miRNA, which indicates that those functions may be weakened although not completely lost. Our findings indicate that HPP is less detrimental to human milk miRNAs than HoP and should be considered in further research on recommended processing procedures for human milk banks.

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Dataset Information

Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

Publications

Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

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