title: Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma.
abstract: BACKGROUND
  Circulating cell-free (ccf) DNA in blood has been suggested as a potential biomarker in many conditions regarding early diagnosis and prognosis. However, misdiagnosis can result due to the limited DNA resources in Biobank's plasma samples or insufficient DNA targets from a predominant DNA background in genetic tests. This study explored several strategies for an efficient DNA extraction to increase DNA amount from limited plasma input.
  
  
  METHODS
  Ccf plasma DNA was extracted with three different methods, a phenol-chloroform-isoamylalcohol (PCI) method, a High Pure PCR Template Preparation Kit method and a method used for single cell PCR in this group. Subsequently, the total DNA was measured by Nanodrop and the genome equivalents (GE) of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantitative assessment of nDNA and mtDNA.
  
  
  RESULTS
  Instead of 400-800 μL (routine input in the laboratory), 50 μLof plasma input enabled the extraction of ccf DNA sufficient for quantitative analysis. Using the PCI method and the kit method, both nDNA and mtDNA could be successfully detected in plasma samples, but nDNA extracted using protocol for single cell PCR was not detectable in 25% of plasma samples. In comparison to the other two methods, the PCI method showed lower DNA purity, but higher concentrations and more GE of nDNA and mtDNA.
  
  
  CONCLUSIONS
  The PCI method was more efficient than the other two methods in the extraction of ccf DNA in plasma. Limited plasma is available for ccf DNA analysis.
authors: Laboratory for Gynecological Oncology, Department of Biomedicine, University Women's Hospital, University of Basel, Basel, Switzerland.
authors: Weijie
authors: W
authors: Chen
authors: Fengfeng
authors: F
authors: Cai
authors: Bei
authors: B
authors: Zhang
authors: Xiao Yan
authors: XY
authors: Zhong
mesh_terms: D001800:Blood Specimen Collection; D002467:Cell Nucleus / Q000737:chemistry*; D019963:Clinical Chemistry Tests / Q000379:methods*; D004247:DNA / Q000097:blood / Q000737:chemistry*; D004272:DNA, Mitochondrial / Q000097:blood; D006801:Humans
chemical_list: D004272:DNA, Mitochondrial; D004247:DNA