Nat Commun. 2025 May 15;16(1):4513. doi: 10.1038/s41467-025-58607-7.
Blood-based extracellular RNA (cell-free RNA; exRNA) biomarkers require validated sample collection, processing, and quantification procedures. No study to date has systematically tested pre-analytical variables affecting transcriptome-wide exRNA analysis. By evaluating their impact on deep transcriptome profiling of microRNAs and mRNAs in blood plasma or serum, we compared ten blood collection tubes, three blood processing time intervals, and eight RNA purification methods. In addition, we assessed interactions among a selected pre-analytical variable set, resulting in 456 extracellular transcriptomes. Blood preservation tubes failed to stabilize exRNA and RNA purification methods differed significantly in performance, causing variations in concentration, detected gene numbers, replicability and observed transcriptome complexity. Critical interactions between tubes, purification methods and time intervals were identified. We provide 11 analytical performance metrics for exRNA quantification methods and put forward recommendations for both users and manufacturers of RNA purification methods and blood collection tubes, collectively, essential groundwork for exRNA-based precision medicine applications.
基于血液的细胞外RNA（无细胞RNA；exRNA）生物标志物需要经过验证的样本采集、处理和定量程序。迄今为止，尚无研究系统地测试影响全转录组exRNA分析的分析前变量。通过评估它们对血浆或血清中微小RNA和信使RNA深度转录组分析的影响，我们比较了十种采血管、三个血液处理时间间隔和八种RNA纯化方法。此外，我们评估了选定分析前变量集之间的相互作用，从而得到了456个细胞外转录组。血液保存管未能稳定exRNA，RNA纯化方法的性能存在显著差异，导致浓度、检测到的基因数量、可重复性和观察到的转录组复杂性出现变化。我们确定了采血管、纯化方法和时间间隔之间的关键相互作用。我们为exRNA定量方法提供了11个分析性能指标，并为RNA纯化方法和采血管的用户及制造商提出了建议，这些共同构成了基于exRNA的精准医学应用的重要基础工作。
