title: A modified extraction method of circulating free DNA for epidermal growth factor receptor mutation analysis.
abstract: OBJECTIVE
  Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol.
  
  
  METHODS
  The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis.
  
  
  RESULTS
  MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (≥ 202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method.
  
  
  CONCLUSIONS
  An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.
authors.affiliation: Department of Oncology, No. 3 People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Haihua
authors.initials: H
authors.lastname: Yuan
authors.forename: Zhong-Zheng
authors.initials: ZZ
authors.lastname: Zhu
authors.forename: Yachao
authors.initials: Y
authors.lastname: Lu
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Wenying
authors.initials: W
authors.lastname: Zhang
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.forename: Guanshan
authors.initials: G
authors.lastname: Zhu
authors.forename: Bin
authors.initials: B
authors.lastname: Jiang
mesh_terms: D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D002725:Chloroform; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000097:blood* / Q000302:isolation & purification*; D066246:ErbB Receptors / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008969:Molecular Sequence Data; D019800:Phenol; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D004273:DNA, Neoplasm; D019800:Phenol; D002725:Chloroform; D066246:ErbB Receptors