Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue
Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue
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DOI：
10.1097/01.pdm.0000213468.91139.2d
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发表时间：
2006-12-01
影响因子：
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通讯作者：
Hewitt, Stephen M.
中科院分区：
文献类型：
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作者：
Chung, Joon-Yong;Braunschweig, Till;Hewitt, Stephen M.
福尔马林固定石蜡包埋（FFPE）组织是显微镜检查后用于组织诊断分析的最常见标本。不仅有大量可用的组织档案，而且FFPE组织仍然是常规临床环境中显微镜检查的最佳制备方法。分子检测，尤其是基于逆转录、聚合酶链反应和表达阵列的检测，作为诊断、预后和预测工具具有巨大的潜力，但需要高质量的RNA。本文优化了一种可靠的FFPE组织RNA提取方法。它是基于高温下的脱胶，加上在65℃下3天的裂解，平均总RNA产量为每1 μ m(3)档案FFPE组织4.5至5.5 pg， 260/280比值在1.80至1.95之间。经生物分析仪分析，提取的RNA的模态片段长度在100 ~ 200 nt之间。虽然RNA片段的模态长度较短，但逆转录和聚合酶链反应能够扩增300 bp范围内的扩增子。RNA预处理后再进行福尔马林固定并不能提高RNA的质量，但确实提高了RNA的产量。我们的方法提高了FFPE组织在分子谱研究中的效用。
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. Not only is there a substantial archive of tissue available, but FFPE tissue remains the best method of preparation for microscopic examination in a routine clinical environment. Molecular assays, especially reverse transcription and polymerase chain reaction and expression array-based assays, offer significant potential as diagnostic, prognostic, and predictive tools, but require high quality RNA. Herein, we have optimized a reliable RNA extraction method for FFPE tissue. It is based on deparaffinization at high temperature coupled with a 3-day lysis at 65 degrees C. The average total RNA yield is 4.5 to 5.5 pg per 1 mu m(3) of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. The extracted RNA has a modal fragment length between 100 and 200 nt by the Bioanalyzer analysis. Although modal lengths of RNA fragments were shorter, reverse transcription and polymerase chain reaction was able to amplify amplicons in range of 300 bp. Pretreatment with RNA later followed by formalin fixation did not result in improving the RNA quality, but did improve RNA yield. Our method improves the utility of FFPE tissue for molecular profiling studies.