Department of Oncology, No. 3 People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
Yonsei Med J. 2012 Jan;53(1):132-7. doi: 10.3349/ymj.2012.53.1.132.
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.
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.
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.
An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.
循环游离 DNA（cfDNA）在血浆中有望成为肿瘤组织 DNA 的替代物。然而，并非所有肿瘤组织 DNA 中的表皮生长因子受体（EGFR）突变都能在匹配的 cfDNA 中检测到，这至少部分归因于 cfDNA 提取方法效率低下。本研究旨在建立一种有效的血浆 cfDNA 提取方案。
通过我们改良的酚-氯仿（MPC）法从非小细胞肺癌（NSCLC）患者中提取的血浆 cfDNA 的产量与 QIAamp MinElute 病毒Spin 试剂盒（Qiagen 试剂盒）作为对照进行比较，采用 Wilcoxon 秩和检验。使用 TaqMan 定量聚合酶链反应（qPCR）检测提取的血浆 cfDNA。同时采用突变富集 PCR（ME-PCR）结合测序和 DxS EGFR 突变检测试剂盒评估提取方法对 EGFR 突变分析的影响。
MPC 法提取的血浆 cfDNA 多于 Qiagen 试剂盒法（p=0.011）。MPC 法提取的 cfDNA 中较长片段（≥202bp）的比例明显高于 Qiagen 试剂盒法（p=0.002）。在 ME-PCR 产物的测序图谱中，MPC 法提取的血浆 cfDNA 中突变峰更高。在 DxS EGFR 突变检测试剂盒结果中，MPC 法提取的血浆 cfDNA 中肿瘤起源 DNA 含量多于 Qiagen 试剂盒法。
提供了一种改良的 MPC 血浆 cfDNA 提取方法，这将有利于 NSCLC 患者的 EGFR 突变分析。
