Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
Virchows Arch. 2012 Feb;460(2):131-40. doi: 10.1007/s00428-011-1184-9. Epub 2012 Jan 22.
Tissues used for clinical diagnostics are mostly formalin-fixed and paraffin-embedded (FFPE) which provides many advantages. However, the quality of the obtained nucleic acids (NA) is reduced and this turns out to be a challenge for further molecular analyses. Although the spectrum of analyses of NA extracted from FFPE tissue has increased, the standard operating procedures for NA isolation from old tissue blocks still need to be improved. Here, we compared the efficiency of different NA extraction methods, using FFPE tissues of variable age and origin, with respect to downstream analyses. Our study showed that the phenol-chloroform isoamyl alcohol (PCI) and the commercial Qiagen protocol yielded samples with highest purity. The PCI protocol delivered the longest amplicons even from samples from the 1970s. We developed a short (1 h) tissue lysis procedure that turned out to be highly time- and cost-effective when DNA quality was tested using single and multiplex PCR. Compared to a 1-day lysis-protocol, the amplicons were only 100 bp shorter. In addition, single-copy genes used in daily routine were successfully amplified from long-term stored FFPE samples following 1-h tissue-lysis. The RNA integrity numbers (RIN) determined on RNA isolated from FFPE tissues indicated degraded RNA; however, all RINs were above the generally agreed threshold of 1.4. We showed that, depending on the purpose of the analysis, NA retrieved from FFPE tissues older than 40 years may be successfully used for molecular analysis.
用于临床诊断的组织大多经过福尔马林固定和石蜡包埋（FFPE），这提供了许多优势。然而，获得的核酸（NA）的质量会降低，这对进一步的分子分析来说是一个挑战。尽管从 FFPE 组织中提取的 NA 分析范围有所增加，但仍需要改进从旧组织块中分离 NA 的标准操作程序。在这里，我们比较了不同 NA 提取方法的效率，使用了不同年代和来源的 FFPE 组织，以进行下游分析。我们的研究表明，酚-氯仿-异戊醇（PCI）和商业 Qiagen 方案从样本中提取出的 NA 纯度最高。即使是来自 20 世纪 70 年代的样本，PCI 方案也能提供最长的扩增子。我们开发了一种简短（1 小时）的组织裂解程序，在使用单重和多重 PCR 测试 DNA 质量时，证明在时间和成本方面非常有效。与 1 天的裂解方案相比，扩增子仅短 100bp。此外，使用单拷贝基因进行日常分析，可以成功地从长期储存的 FFPE 样本中扩增出来，而无需进行 1 小时的组织裂解。从 FFPE 组织中分离的 RNA 的 RNA 完整性数值（RIN）表明 RNA 降解；然而，所有 RIN 均高于通常约定的 1.4 阈值。我们表明，根据分析的目的，可成功地从超过 40 年的 FFPE 组织中提取 NA ，用于分子分析。
