Basic research on the appropriate storage conditions for cell-free DNA: Attempts towards standardization of liquid biopsy

Abstract

Comprehensive genomic profiling method typically uses tissue samples to analyze cancer-related genes via next-generation sequencing. Liquid biopsy using blood cell-free DNA (cfDNA) has recently gained attention. However, standardization of procedures from blood collection to analysis remains challenging, particularly due to the unknown effects of collection tube type and storage conditions on cfDNA quality. In this study, blood samples were collected from 10 healthy individuals in three types of tubes: ethylenediaminetetraacetic acid (EDTA)-2Na, EDTA-2K, and cfDNA tubes. cfDNA was extracted from the plasma, and total DNA concentration was measured via fluorescence assay. All samples were stored at –80°C and analyzed at different time points (within 7 days and 1, 3, and 6 months), and cfDNA band patterns were examined. Notably, no significant differences in total DNA yield were observed among all tube types. Moreover, cfDNA band patterns were consistent across all tube types and storage durations. Therefore, both EDTA tubes can replace cfDNA tubes for cfDNA extraction. Furthermore, cfDNA quality was maintained for up to 6 months under –80°C storage conditions. Overall, this study provides critical insights for the standardization of cfDNA analysis in liquid biopsy, facilitating the design of more reliable and reproducible workflows in clinical and research settings.