Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.
Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.
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DOI：
10.1371/journal.pone.0176280
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Esumi M
中科院分区：
文献类型：
--
作者：
Einaga N;Yoshida A;Noda H;Suemitsu M;Nakayama Y;Sakurada A;Kawaji Y;Yamaguchi H;Sasaki Y;Tokino T;Esumi M
用于病理诊断的福尔马林固定石蜡包埋（FFPE）组织对于研究癌症基因组学很有价值。特别是，通过组织病理学结合 FFPE 组织切片免疫组织化学 (IHC) 确定的靶细胞的激光捕获显微切割，可以通过下一代测序 (NGS) 对癌症中发生的遗传事件进行精确分析。其结果是癌症诊断病理学工具的新策略：“微观基因组学”。为了更方便、更精确地进行微观基因组学，我们通过系统分析揭示了 FFPE DNA 与配对冷冻组织 DNA 相比的以下三个细节。 1) 最佳质量的 FFPE DNA 是通过用 10% 中性缓冲福尔马林组织固定 1 天，然后将组织裂解物在 95°C 下热处理 30 分钟来获得的。 2) FFPE 组织的 IHC 染色使 FFPE DNA 的数量和质量降低至四分之一，抗原修复（120°C 15 分钟，pH 6.0）是造成这种下降的主要原因。 3) 按本文所述制备的 FFPE DNA 足以用于 NGS。对于非突变组织样本，FFPE DNA 和配对冷冻组织 DNA 的 NGS 进行精确比较并随后进行验证表明，在 FFPE 制备过程中不会发生人为突变。这些结果表明，如果应用适当的固定和验证条件，即使用于常规临床诊断的 FFPE 组织也可用于获得可靠的 NGS 数据。
Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.