Pre-analytical considerations of the recovery and downstream analysis of circulating nucleic acids

Establishing robust pre-analytical quality standards is essential to improve the reliability and reproducibility of liquid biopsy assays based on circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA). Variability in sample collection, processing, and storage can substantially influence nucleic acid recovery and downstream biomarker analyses. In this study, we developed a structured framework to evaluate key pre-analytical variables affecting cfDNA and cfRNA workflows. The framework was applied to assess blood collection tubes, nucleic acid extraction methods, and major sources of pre-analytical variation, including storage conditions, transportation, freeze–thaw cycles, and underfilled blood collection tubes. Under the conditions evaluated, the Streck RNA Complete Blood Collection Tube (BCT) supported preservation of cfDNA and cfRNA and was associated with lower levels of cellular contamination compared with K3-EDTA tubes. Among the extraction methods evaluated, the MagMAX cfDNA and Zymo cfRNA kits demonstrated high nucleic acid recovery and compatibility with scalable workflows. Delayed processing, repeated freeze–thaw cycles, and reduced tube fill volumes were associated with decreased nucleic acid yield, increased cellular contamination, and greater variability in downstream measurements. Overall, the proposed framework provides a systematic approach for evaluating pre-analytical factors that influence liquid biopsy performance under controlled experimental conditions. While the findings are based on the specific products and workflows examined in this study, the framework may support workflow standardization and provide a basis for assessing emerging technologies in liquid biopsy research and translational applications.