Extraction of Cell-Free DNA: Evaluation of Efficiency, Quantity, and Quality

Cell-free DNA (cfDNA) serves as a valuable biomarker for early disease detection and monitoring. However, the use of cfDNA for analysis faces challenges owing to general low but variable abundance and fragmentation. Preanalytical factors, including cfDNA extraction, impact cfDNA quality and quantity. Efficient and robust cfDNA extraction is essential for reliable results in downstream applications, and various commercial extraction methods exist, each with trade-offs. To aid researchers and clinicians in choosing the proper cfDNA extraction method, manual, semiautomated, and automated methods were evaluated, including the QIAamp Circulating Nucleic Acid Kit (manual and QIAcube), QIAamp MinElute ccfDNA Kit (QIAcube), and QIAsymphony DSP Circulating DNA Kit (QIAsymphony). For each extraction method, cfDNA was extracted on two separate days, using samples obtained from 18 healthy donors. This study assessed extraction efficiency, quantity, and quality using droplet digital PCR and TapeStation. The QIAamp Circulating Nucleic Acid Kit, both manual and semiautomated, outperformed the QIAamp MinElute ccfDNA Kit (QIAcube) and QIAsymphony DSP Circulating DNA Kit (QIAsymphony), showing higher recovery rates and cfDNA quantity. All methods were reproducible, with no day-to-day variability and no contamination by high-molecular-weight DNA. The QIAamp Circulating Nucleic Acid Kit offers high yield without compromising quality. Implementation of the method should consider specific study and clinical needs, taking into account each method's advantages and limitations for optimal outcomes.