PO.PR01.03 · 预防研究
环境温度下cfRNA的保存：标准化提取揭示多分析物采集管中更优的稳定性
AmbientcfRNApreservation: standardized extraction reveals superior stability in a multi-analyte tube
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：液体活检读数对分析前变异性高度敏感：细胞裂解和核酸酶活性可增高背景、抑制稀有信号并损害重复性。因此，在采集时同时稳定多个靶标，并配合受控的提取工作流程至关重要。我们评估了两种血液采集管——一种设计用于多分析物稳定性的单管基质——采用基于磁珠的化学方法，重点关注cfRNA信号完整性以及与下游RT-qPCR的兼容性。
方法：将来自健康供者的全血采集到Tube-A（TAG FLEX-LB™）和Tube-B（Streck Nucleic Acid BCT）中，等分样本按每mL固定浓度加入了来自H441细胞（KRAS G12V）的纯化总RNA。采集管在约21-25℃下储存，并在T0和T7进行处理。使用二氧化硅磁珠Revolution cfTNA Max 20试剂盒分离cfRNA。终点指标：针对KRAS G12V的等位基因特异性RT-qPCR（Ct；ΔCt = T7-T0；扩增效率和回收率）、溶血替代指标hsa-miR-16（ΔCt），以及RNA完整性（RIN；18S/28S）。
统计学：采用配对检验并给出95%置信区间。
结果：两种采集管采用同一提取工作流程时，KRAS G12V在T0均稳定扩增。在室温放置7天后，Tube-A显示出极小的降解、保持了扩增效率并维持了高回收率。Tube-B则表现出更大程度的降解以及回收效率的下降。Tube-A的溶血信号更低：第7天的miR-16丰度低于Tube-B。Tube-A中RNA完整性保持稳定，而Tube-B中则下降。Tube-A的检测QC合格率更高。
结论：在真实的7天环境温度放置条件下，当稳定化充分时，cfRNA靶标仍可定量。在这项头对头研究中，采用相同的提取工作流程，多分析物稳定化采集管相较于另一种采集管更好地保留了KRAS-G12V的可检测性、显示出更低的溶血，并维持了RIN/18S-28S指标。这些发现支持cfRNA工作流程，并强调了对涵盖保存和提取两方面的标准化分析前流程的需求。
查看英文原文 English abstract
Introduction: Liquid-biopsy readouts are highly sensitive to pre-analytical variability: cellular lysis and nuclease activity can inflate background, suppress rare signals, and impair reproducibility. Concurrent stabilization of multiple targets at the time of collection, paired with a controlled extraction workflow, is therefore critical. We assessed two blood-collection tubes-a single-tube matrix designed for multi-analyte stability-using the magnetic bead-based chemistry, focusing on cfRNA signal integrity and compatibility with downstream RT-qPCR.
Methods: Whole blood from healthy donors was drawn into Tube-A (TAG FLEX-LB™) and Tube-B (Streck Nucleic Acid BCT) aliquots were spiked with purified total RNA from H441 cells (KRAS G12V) at a fixed concentration per mL. Tubes were stored at ~21-25 °C and processed at T0 and T7. cfRNA was isolated using silica-magnetic bead Revolution cfTNA Max 20 Kit. Endpoints: allele-specific RT-qPCR for KRAS G12V (Ct; ΔCt = T7-T0; amplification efficiency and recovery), hemolysis proxy hsa-miR-16 (ΔCt), and RNA integrity (RIN; 18S/28S).
Statistics: paired tests with 95% Confidence Intervals.
Results: With one extraction workflow across both tubes, KRAS G12V amplified consistently at T0. After 7 days at room temperature, Tube-A showed minimal degradation, preserved efficiency, and maintained high recovery. Tube-B exhibited greater degradation and reduced efficiency in recovery. Hemolysis signal was lower in Tube-A: Day-7 miR-16 abundance was lower than Tube-B. RNA integrity remained stable in Tube-A but declined in Tube-B. Assay QC pass rate were higher for Tube-A.
Conclusions: Under a realistic 7-day ambient hold, cfRNA targets remained quantifiable when stabilization was adequate. In this head-to-head study, the multi-analyte stabilizing tube better preserved KRAS-G12V detectability, showed lower hemolysis, and maintained RIN/18S-28S metrics relative to a different tube, using an identical extraction workflow. These findings support cfRNA workflows and underscore the need for standardized pre-analytics-spanning both preservation and extraction.
利益披露 Disclosure
E. Medina, None..
T. Baker, None.