title: 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 microl 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.
authors.affiliation: Academic Unit of Clinical Oncology, University of Sheffield, Sheffield, UK.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Xue
authors.forename: M Dawn
authors.initials: MD
authors.lastname: Teare
authors.forename: Ingunn
authors.initials: I
authors.lastname: Holen
authors.forename: Yong M
authors.initials: YM
authors.lastname: Zhu
authors.forename: Penella J
authors.initials: PJ
authors.lastname: Woll
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods; D004273:DNA, Neoplasm / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis*; D010636:Phenols; D011379:Prognosis; D011933:Reagent Kits, Diagnostic
chemical_list: D004273:DNA, Neoplasm; D010636:Phenols; D011933:Reagent Kits, Diagnostic