Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations.
Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations.
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DOI：
10.1371/journal.pone.0167969
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Tfelt-Hansen J
中科院分区：
文献类型：
--
作者：
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
microRNA（miRNA）作为非侵入性诊断、预后和预测生物标志物以及治疗靶点的潜力最近已被认识到。以前的研究强调了所用方法一致性的重要性，但据我们所知，没有研究系统地描述了关于miRNA作为血液生物标志物的样品制备和储存方法。本研究的目的是研究血液中miRNA在各种相关临床和研究条件下的稳定性：不同收集管，不同温度下储存，物理干扰以及系列冻融循环。从12名健康供体采集血样，置于含有抗凝剂（包括EDTA、柠檬酸盐和肝素锂）的不同采集管中，以及血清采集管中。通过测量不同条件下miR-1、miR-21和miR-29 b的表达变化来评估miRNA稳定性：全血的不同处理时间（长达72小时（h））、长期储存（在-80 ℃下储存9个月）、物理干扰（1和8 h）以及一系列冻融循环（1和4次）。不同的收集管显示miR-1、miR-21和miR-29 b的浓度相当。发现具有肝素锂的管不适合于miRNA定量。在室温下，全血中的miRNA水平稳定至少24小时，而分离的组分在24小时内确实显示出变化。在室温下孵育全血72小时后，miR-21和miR-29 b水平有显著变化（两者均为p<0.01）。分离血浆中miR-1和miR-21的水平在物理干扰8 h后降低，血清全血中miR-1的水平在物理干扰1 h后未观察到变化。在-80 ° C下储存样品显著延长了miRNA的稳定性，然而，长期储存（9个月）的全血样品中的miRNA水平显著改变，这与血浆样品形成对比，其中发现miR-21或miR-29 b水平稳定。重复（n = 4）冻融循环导致血浆和血清样品中的miRNA浓度显著降低。这项研究强调了在测量循环miRNA时，适当和系统的样本收集和制备的重要性，例如，在临床试验中。我们证明，收集管的类型，样品的制备，处理和储存应标准化，以避免混淆变量影响结果。
The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles. Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times). Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples. This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.