OPTIMIZATION OF EXTRACTION AND PCR AMPLIFICATION OF RNA EXTRACTS FROM PARAFFIN-EMBEDDED TISSUE IN DIFFERENT FIXATIVES
OPTIMIZATION OF EXTRACTION AND PCR AMPLIFICATION OF RNA EXTRACTS FROM PARAFFIN-EMBEDDED TISSUE IN DIFFERENT FIXATIVES
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DOI：
10.1016/0166-0934(93)90076-4
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发表时间：
1993-07-01
影响因子：
3.1
通讯作者：
ZAKI, SR
中科院分区：
文献类型：
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作者：
KOOPMANS, M;MONROE, SS;ZAKI, SR
建立了一种从石蜡包埋的组织切片中快速有效地分离RNA的方法，用于随后的PCR分析。该方法基于在高摩尔浓度胍盐存在下RNA与酸处理的玻璃珠的结合。它可以在一个小时内完成，并避免了脱蜡和苯酚/氯仿萃取的需要。测试了各种固定剂和固定时间的影响，并使用长度范围为82至507 bp的肌动蛋白mRNA片段的扩增来证明提取物中RNA的存在。通过研究逆转录聚合酶链反应扩增（RT-PCR）模板的质量，使用病毒感染和模拟感染的石蜡包埋细胞团作为模型，该方法进行了比较，以现有的提取技术。细胞和病毒RNA的PCR扩增是成功的RNA分离使用所有的提取技术，虽然玻璃珠的方法是最好的，因为它的简单和快速。用福尔马林固定的标本被认为是适合PCR，但最好的结果与丙酮固定的石蜡包埋材料。组织切片的脱蜡对RNA提取的产量和质量没有影响，并且使用凝胶过滤进一步纯化提取物没有改善结果。在优化方案后，使用轮状病毒感染的细胞沉淀来证明dsRNA的提取和扩增是可能的。从模型系统研究中获得的信息用于从存档肠道材料中提取圆环病毒和轮状病毒RNA。这些数据表明，石蜡包埋的档案组织可用于RT-PCR分析，增加了一个重要的技术，诊断病理学和回顾性研究。
A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.