Heikal Nahla, Nussenzveig Roberto H, Agarwal Archana M
*Department of Pathology †Associated Regional University Pathologists (ARUP) Laboratories, University of Utah, Salt Lake City, UT.
Appl Immunohistochem Mol Morphol. 2014 Sep;22(8):623-6. doi: 10.1097/PAI.0b013e3182a77bfe.
Extracting DNA from formalin-fixed paraffin-embedded (FFPE) archival samples remains difficult. Successful polymerase chain reactions (PCR) with DNA extracted from FFPE samples is still very low. We extracted DNA from 12 recent and old archival FFPE bone marrow trephine biopsies by use of a simple protocol on the basis of deparaffinization with molecular biology-grade mineral oil followed by DNA extraction with the Qiagen FFPE kit. Comparison of this deparaffinization method with standard protocols, for example, xylene or Hemo-D with subsequent rehydration using graded ethanols, was investigated. The quality and quantity of extracted DNA were tested by a combination of ultraviolet spectroscopy, analysis on a Caliper LabChip GX, and real-time PCR combined with high-resolution melt analysis. Highest quality PCR-amplifiable DNA was obtained by deparaffinization with mineral oil, whereas more variable results were obtained for the other 2 deparaffinization procedures. This result was confirmed by real-time PCR and high-resolution melt analysis. Besides improvements in the quality of extracted DNA, use of mineral oil for deparaffinization has the added benefit of decreased time (20 vs. 75 min) and a significant reduction of hands-on labor (1 step vs. multiple hands-on centrifugation and decanting steps).
从福尔马林固定石蜡包埋(FFPE)存档样本中提取DNA仍然具有挑战性。使用从FFPE样本中提取的DNA成功进行聚合酶链反应(PCR)的比例仍然很低。我们采用一种简单的方案从12份近期和陈旧的存档FFPE骨髓环钻活检样本中提取DNA,该方案基于用分子生物学级矿物油进行脱石蜡处理,随后使用Qiagen FFPE试剂盒进行DNA提取。研究了这种脱石蜡方法与标准方案(例如二甲苯或Hemo-D,随后使用梯度乙醇进行复水)的比较。通过紫外光谱、Caliper LabChip GX分析、实时PCR结合高分辨率熔解分析相结合的方法对提取的DNA的质量和数量进行了检测。通过用矿物油进行脱石蜡处理可获得质量最高的可用于PCR扩增的DNA,而其他两种脱石蜡方法得到的结果则更具变异性。实时PCR和高分辨率熔解分析证实了这一结果。除了提高提取的DNA的质量外,使用矿物油进行脱石蜡处理还有减少时间(20分钟对75分钟)和显著减少人工操作(1步对多步人工离心和倾析步骤)的额外好处。