Cell-free DNA quality and quantity assessment ─ A method comparison

Workflow standardization is required for successful use of circulating cell-free DNA (ccfDNA) as a cancer research analyte. The international technical specification for isolation of ccfDNA (CEN/TS 16835-3:2015) focuses on preanalytical factors like preventing cellular DNA release by using blood stabilization tubes and dedicated isolation procedures. In addition, accurate determination of ccfDNA quality and quantity is required for downstream analyses like NGS, quantitative or digital PCR (qPCR/dPCR). This research study compares common methods for quality control of ccfDNA from unstored and stored blood and different tube processing conditions.

Blood from 8 donors was collected in EDTA and PAXgene Blood ccfDNA Tubes (PAXgene)*. Paired tubes were processed directly or after storage for 7 days at 25°C. CcfDNA was isolated using the QIAamp Circulating NA Kit, the QIAsymphony Circulating DNA Kit (EDTA) or the QIAsymphony PAXgene Blood ccfDNA Kit* (PAXgene). All 64 samples were analyzed for ccfDNA quality and/or quantity with QC methods based on fluorescence, automated electrophoresis and PCR.

Differences in ccfDNA quality and quantity were observed between QC methods. CcfDNA fragment distribution was shown for electrophoretic methods. Based on the sample characteristics and required QC parameters, different technologies are suitable for sample assessment. The release of genomic DNA over time indicated the need for stabilization and an accurate quantification method for optimal workflow control.

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^┼For Research Use Only. Not for use in diagnostic procedures.