Comparison of 7 Methods for Extracting Cell-Free DNA from Serum Samples of Colorectal Cancer Patients

Abstract

The presence of cell-free DNA of tumor origin in serum or plasma of cancer patients (1) has triggered numerous studies to explore the diagnostic and prognostic potential of circulating DNA. This DNA, present only in minute concentrations in plasma or serum, is highly fragmented (2), a condition that often leads to substantial loss of DNA of small sizes in the course of DNA isolation. The lack of consensus regarding which extraction method is better for the efficient capture of such DNA might be partially responsible for the large disparities in the literature, which are reflected in reports of total concentrations of plasma or serum DNA alone (3) or DNA integrity measurement as a diagnostic or prognostic tool. The ability to detect mutated v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog ( KRAS ) DNA in serum has been reported to vary with the chosen DNA isolation methods (4). We evaluated in parallel 7 isolation approaches (Table 1⇓ ) by extracting cell-free DNA from 12 pooled sera obtained from 67 colorectal cancer patients and grouped on the basis of TNM tumor staging [tumor extent (T), spread to lymph nodes (N), and metastasis (M)].1 The approaches we evaluated involved diverse strategies for DNA isolation. The experimental set-up involved DNA isolation from a 2-mL aliquot of serum in duplicate followed by DNA quantification by the fluorescent Quant-iT dsDNA HS assay (Invitrogen) and a Taqman real-time PCR (rPCR) technique on the cadherin 1, type 1, E-cadherin (epithelial) ( CDH1 ) gene. To exclude false results in the CDH1 amplification due to …