Size distribution of cell-free DNA from plasma and urine depending on stabilization and analysis method

Introduction: Cell-free DNA (cfDNA) is a key analyte for liquid biopsy samples. Due to a short half-life of around 15min, very low concentrations of cfDNA, combined with a high fragmentation, is found in samples. Calculated size profiles, therefore, strongly depend on sample collection as well as the analysis method used, which often results in a biased analysis of the ‘true’ cfDNA size. Here, different factors such as stabilization and the extraction method which impact the size profile are analyzed in detail. Additionally, we considered the impact of the analysis method used.

Methods: Plasma and urine were subjected to different stabilization conditions. CfDNA was then extracted using a manual column-based method and an automated bead-based method on the QIAsymphony SP instrument using 10 ml input volume. The size profile of the extracted cfDNA was analyzed by a gel-based size separation and subsequent qPCR.

Results: A significant shift in the size profile of cfDNA was determined within a few hours of storage with large differences which depended on the source of cfDNA, plasma, and urine. The extraction method used also had a strong impact on the extracted size profile, in particular, for small cfDNA fragments around 100bp. In this context, the analysis method used defines the measured cfDNA, e.g. focus on total cfDNA - ssDNA and dsDNA - or focus on the recovery of larger cfDNA fragments.

Conclusions:

Standardization of pre-analytical workflows as well as usage of downstream assays should be considered to get a complete picture of the released DNA into body fluids.

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