从1-20 mL输入量中整合回收DNA/RNA可在液体活检模型中实现敏感的KRAS G12V检测

Introduction: Liquid-biopsy applications increasingly need concurrent access to tumor-derived DNA and RNA, sensitivity at low variant abundance, and support for larger input volumes. Separate cfDNA/cfRNA workflows add hands-on time and can limit sensitivity. We evaluated a single-extraction total nucleic acid (TNA) workflow designed to co-recover circulating DNA and RNA across inputs ranging from 1 to 20 mL. Methods: Conditioned media from KRAS-G12V-positive H441 cells were pooled, clarified, and filtered to form a uniform tumor-conditioned matrix. Replicate extractions (n = 5 per condition) were performed at 1, 5, 10, and 20 mL using the same silica-magnetic-bead TNA chemistry and a single fixed-volume eluate. Eluate was split post-extraction for DNA (Qubit HS DNA; KRAS-G12V qPCR) and RNA (Qubit HS RNA; RT-qPCR). Metrics included total yield, KRAS-G12V copies/mL, allele fraction, and coefficient of variation (CV). Rare-event performance was tested using a dilution series (100%, 20%, 5%, 1%, 0.2% KRAS-G12V) extracted at 20 mL (n = 5 per level). Fragment integrity for 1 mL vs 20 mL inputs was evaluated by high-sensitivity capillary electrophoresis. Results: KRAS-G12V copies/mL scaled linearly with input volume for both DNA- and RNA-derived signal (R²≥0.98 across 1-20 mL; within-volume CV≤15%). Increasing input reduced allele-fraction variance, improving quantitative precision without changing chemistry or handling. Split-eluate analysis showed strong concordance between DNA and RNA KRAS-G12V copies/mL across volumes, demonstrating that one extraction supports both genomic (mutation) and transcriptional readouts. In dilution experiments at 20 mL, KRAS-G12V was consistently detected down to 0.2% variant fraction. Fragment analysis confirmed cf-like size profiles (~160-180 bp) with minimal high-molecular-weight carryover at all inputs. Conclusions: A single-eluate TNA workflow co-recovers DNA and RNA from up to 20 mL input, yields linear signal scaling, preserves cf-like integrity, and maintains detection at low simulated tumor fractions. Larger inputs not only boost recovery but also tighten allele-fraction precision. These data support high-volume, integrated DNA/RNA liquid biopsy workflows for applications such as minimal residual disease monitoring and longitudinal response assessment.