Katayama Erryk S, Hue Jonathan J, Loftus Alexander W, Ali Semmer A, Graor Hallie J, Rothermel Luke D, Londin Eric, Zarei Mehrdad, Winter Jordan M
Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA.
Noncoding RNA Res. 2025 Aug 8;15:132-141. doi: 10.1016/j.ncrna.2025.08.001. eCollection 2025 Dec.
To verify the stability and reliability of circulating microRNA (miRNA) profiles in plasma and serum under different processing and storage conditions to inform future applications to circulating biomarker analyses.
The development of blood-based methods for early disease detection has become increasingly desirable across various medical fields. RNA profiles have been investigated but have been a challenge due to rapid degradation of the analyte by ubiquitous RNases. miRNAs are short, non-coding regulatory RNAs that are believed to be more stable under certain conditions, large in number, and specific to cell type and disease state. Thus, circulating miRNA profiles hold significant promise as diagnostic biomarkers for a range of conditions, including cancer, autoimmune, liver, neurological, metabolic, and cardiovascular diseases. However, to realize their full potential in clinical applications, it is crucial to thoroughly characterize the stability of miRNAs under various blood collection, processing, and storage conditions prior to their investigation and large-scale application in disease-specific biomarker discovery studies.
Plasma or serum were extracted from whole blood of healthy volunteers. Samples were stored at different temperatures (4 °C or 25 °C, room temperature) for varying periods (0-24 h) to mimic possible delays in processing encountered in routine clinical settings. miRNA was extracted and profiles were assessed with RT-qPCR or small RNA-sequencing techniques.
Mean Cq values of specific miRNAs, such as miR-15b, miR-16, miR-21, miR-24, and miR-223, remained consistent between 0 and 24 h when serum and plasma were stored on ice. Minimal changes were observed in mean Cq values over 24 h when serum was left at room temperature as well. Similar trends were observed when miRNAs from plasma were analyzed. Small-RNA sequencing detected approximately ∼650 different miRNA signals in plasma, with over 99 % of the miRNA profile unchanged even when blood draw tubes were left at room temperature for 6 h prior to processing.
These data demonstrate remarkable stability of miRNAs over time, which should withstand variability in handling and processing that can occur with routine clinical lab draws. Considering the large diversity of miRNAs, this analyte class should be thoroughly investigated as a non-invasive biomarker of diverse disease states.
验证在不同处理和储存条件下，血浆和血清中循环微RNA（miRNA）谱的稳定性和可靠性，为其在循环生物标志物分析中的未来应用提供参考。
基于血液的早期疾病检测方法的开发在各个医学领域中变得越来越迫切。RNA谱已被研究，但由于普遍存在的核糖核酸酶会迅速降解分析物，这一直是一个挑战。miRNA是短的非编码调节RNA，据信在某些条件下更稳定，数量众多，并且对细胞类型和疾病状态具有特异性。因此，循环miRNA谱作为一系列疾病的诊断生物标志物具有巨大潜力，包括癌症、自身免疫性疾病、肝脏疾病、神经疾病、代谢疾病和心血管疾病。然而，为了在临床应用中充分发挥其潜力，在将其用于疾病特异性生物标志物发现研究的调查和大规模应用之前，全面表征miRNA在各种血液采集、处理和储存条件下的稳定性至关重要。
从健康志愿者的全血中提取血浆或血清。将样品在不同温度（4°C或25°C，室温）下储存不同时间（0 - 24小时），以模拟常规临床环境中可能遇到的处理延迟。提取miRNA并使用RT-qPCR或小RNA测序技术评估其谱。
当血清和血浆在冰上储存时，特定miRNA（如miR-15b、miR-16、miR-21、miR-24和miR-223）的平均Cq值在0至24小时之间保持一致。当血清在室温下放置24小时时，平均Cq值也观察到最小变化。分析血浆中的miRNA时也观察到类似趋势。小RNA测序在血浆中检测到约650种不同的miRNA信号，即使采血试管在处理前在室温下放置6小时，超过99%的miRNA谱也没有变化。
这些数据表明miRNA随时间具有显著的稳定性，能够经受常规临床实验室采血过程中可能出现的处理和操作变化。考虑到miRNA的多样性，应将这类分析物作为多种疾病状态的非侵入性生物标志物进行全面研究。
