Optimizing the yield and utility of circulating cell-free DNA from plasma and serum

Background: Cell-free DNA (CFDNA) in the plasma/serum of patients with cancer demonstrates tumour-associated genetic alterations. offering possibilities for diagnosis, prognostication and disease monitoring. There is wide variation in the reported levels of CFDNA, associated with different methods used to collect, process and analyze blood samples. We therefore evaluated different aspects of laboratory protocols for the processing and purification of CFDNA in clinical studies.

Methods: We evaluated and compared the QlAamp kit and a Triton/Heat/Phenol protocol (THP) for CFDNA purification. Total CFDNA was quantified by PicoGreen assay and SYBR-Green real-time PCR assay was used to amplify specific genes to estimate the efficiency of different protocols.

Results: The efficiency of DNA extraction was 18.6% using the standard QlAamp protocol and 38.7% using the THP method (p<0.0001, unpaired t-test). A modified QlAamp protocol that included a proteinase incubation stage and elution volumes up to 300 mu l increased DNA yields, but was not as good as the THP method.

Conclusions: Blood samples should be kept at/or below room temperature (18 degrees C-22 degrees C) for no more than 2 h before plasma separation by double-spin. Because of its higher efficiency, low-cost and good-quality products, the THP protocol is preferred for extraction of CFDNA.