Toward a Robust Cell-free DNA Isolation Protocol for Next-Generation Sequencing Applications in a Clinical Molecular...

Cell-free (cf) DNA, released from apoptotic and necrotic cells into bodily fluids, is a noninvasive source of genetic information for disease prediction, diagnosis, and monitoring. However, its low abundance makes cfDNA highly susceptible to various preanalytical influences, potentially increasing high-molecular-weight or genomic DNA, compromising downstream cfDNA analysis. This study evaluated the impact of different cfDNA-stabilizing blood collection tubes (Cell-Free DNA BCT, Streck; S-Monovette cfDNA Exact, Sarstedt) stored at room temperature for 1, 5, or 10 days prior to plasma isolation using different isolation methods (magnetic bead based or silica column based) on cfDNA stability and yield. DNA quantity and quality were assessed by fluorometric quantification, automated fragment analysis, and gene-specific real-time quantitative PCR. Streck-based workflows maintained stable cfDNA yields and characteristic mononucleosomal fragmentation profiles across all storage times. In contrast, Sarstedt tubes showed reduced cfDNA concentrations after 5 days and a pronounced increase at 10 days, accompanied by high-molecular-weight DNA patterns consistent with white blood cell lysis. These trends were largely independent of the extraction method. Overall, the results demonstrate that blood collection tube chemistry critically influences cfDNA integrity during delayed processing. The Streck tubes, particularly when combined with the silica column-based isolation method, provided the most robust and reproducible workflow for routine molecular diagnostics, whereas the Sarstedt tubes produced physiologically implausible results after extended storage.