Evaluation of formalin-free tissue fixation for RNA and microRNA studies
Evaluation of formalin-free tissue fixation for RNA and microRNA studies
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DOI：
10.1016/j.yexmp.2011.05.007
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发表时间：
2011-10-01
影响因子：
3.6
通讯作者：
Popper, Helmut H.
中科院分区：
文献类型：
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作者：
Arzt, Lisa;Kothmaier, Hannelore;Popper, Helmut H.
将FineFix、RCL-2和HOPE三种无福尔马林固定剂与常用的福尔马林固定的肺癌组织标本进行了比较，并评价了它们对RNA和microRNA质量、数量和完整性的影响。为了确定不依赖固定剂的RNA和miRNA的提取方法，对两种商业RNA提取试剂(Qiagen的RNasy FFPE和Ambion的RecoverAll(TM)核酸分离试剂盒)进行了测试和优化。两个选定的miRNAs通过TaqMan microRNA分析进行了定量。优化的Qiagen‘s Kit的RNA提取方案在所有固定剂的RNA质量和完整性方面都产生了相似的结果。福尔马林的RNA产量最高，FineFix的平均miRNA比率最高。在FineFix、福尔马林和RCL2固定的组织中检测到小于500个碱基的RNA片段；在三分之一的样本中，HOPE是唯一显示长片段的固定剂。我们的发现表明，在RNA研究中，无福尔马林固定剂总体上并不优越。通过我们优化的RNA提取方案，不存在高质量地提取大量RNA的困难。根据所获得的质量，可以进行定量实时PCR分析，而不会产生任何负面影响。对于被测试的固定剂也可以得到类似的结果，因此没有固定剂似乎代表了组织固定的新的“黄金标准”。(C)2011 Elsevier Inc.保留所有权利。
FineFix, RCL-2 and HOPE, three formalin-free fixatives, were compared to the common used formalin fixed tissue samples of lung cancer and were evaluated for their effects on quality, quantity and integrity of RNA and microRNA.Two commercially available RNA extraction Kits (RNeasy FFPE by Qiagen and RecoverAll (TM) Nucleic Acid Isolation by Ambion) were tested and optimized in order to determine an extraction protocol for RNA as well as miRNA independent of the fixative. Two selected miRNAs were quantified via TaqMan MicroRNA assays. The optimized RNA extraction protocol for Qiagen's Kit leads to similar results for RNA quality and integrity for all fixatives. Highest RNA yield was obtained for formalin and the highest average miRNA ratio was found for FineFix. RNA fragments smaller than 500 bases were detected in FineFix, formalin and RCL2 fixed tissues; HOPE was the only fixative showing long fragments in one third of the samples. Our findings demonstrate that formalin-free fixatives are in general not superior for RNA studies. With our optimized RNA extraction protocol, there is no difficulty in extracting great amounts of RNA with high quality. According to the quality obtained, quantitative real-time PCR analysis can be performed without any negative impact. Similar results can be achieved for the tested fixatives and therefore no fixative seems to represent a new "gold-standard" for tissue fixation. (C) 2011 Elsevier Inc. All rights reserved.