Comparison of methods for the isolation and high quality assessment of cell-free DNA from blood plasma

Background: Analysis of cell-free tumor DNA (ctDNA) has shown to have great potential as “liquid biopsy” in oncological diagnostics for treatment stratification, disease monitoring and early detection of tumor recurrence. However, preanalytical sample handling and preparation are crucial for high quality results. This includes sample collection, transportation, centrifugation and storage, DNA extraction and library preparation for NGS.

Study Aim: To compare five commercially available cfDNA manual extraction kits and one automated method and evaluate the yield the purity and the size of cfDNA obtained from human plasma samples.

Materials and Methods: cfDNA was manually and automatically extracted from 1 ml plasma of 10 donors using affinity column and magnetic-bead-based assays from five manufacturers: QIAamp MinElute ccfDNA Mini Kit (Qiagen), QIAamp Circulating Nucleic Acid kit (Qiagen), MagMAX Cell-Free DNA Isolation Kit (ThermoFisher Scientific), NucleoSpin Plasma XS (Macherey.Nagel), cfPure^TM Cell-Free DNA Extraction Kit (BioChain) and MagNa Pure 24 Total NA Isolation Kit with the cfDNA Buffer Set. DNA extracts were analysed by fluorometric quantitation (Qubit, dsDNA High-Sensitivity assay) and by Agilent Bioanalyzer (High Sensitivity DNA Kit) to assess concentrations, DNA fragment sizes and genomic DNA contamination.

Results: There were considerable differences regarding the DNA yield obtained by the various extraction kits. Similarly, the purity of the samples and contamination by gDNA varied considerably when using different extraction kits. In addition, purity was influenced by other preanalytical factors such as double centrifugation and storage conditions. Two of the six tested kits performed very well in terms of DNA yield and purity and can be recommended for DNA extraction. Other assays show strengths and limitations but may be appropriate for special applications of the user.

Conclusion: Two cfDNA kits enable highly sensitive and pure cfDNA extraction from plasma as an essential precondition for downstream PCR and NGS analyses.