AmbientcfRNApreservation: standardized extraction reveals superior stability in a multi-analyte tube

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.