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

Abstract

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 QIAamp 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 QIAamp protocol and 38.7% using the THP method ( p < 0.0001, unpaired t-test). A modified QIAamp protocol that included a proteinase incubation stage and elution volumes up to 300 μl increased DNA yields, but was not as good as the THP method. Conclusions Blood samples should be kept at/or below room temperature (18 °C–22 °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.