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一种基于 HPLC 的固定后 RNA 研究新方法

A Novel HPLC-Based Method to Investigate on RNA after Fixation.

机构信息

DSM-Department of Medical Sciences, University of Trieste, 34149 Trieste, Italy.

Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy.

出版信息

Int J Mol Sci. 2020 Oct 13;21(20):7540. doi: 10.3390/ijms21207540.

DOI:10.3390/ijms21207540
PMID:33066070
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7588918/
Abstract

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

摘要

从固定和石蜡包埋组织中分离的 RNA 广泛用于生物医学研究和分子病理学诊断。在本研究中，我们建立了一种基于高效液相色谱 (HPLC) 的方法来研究不同固定剂对 RNA 的影响。通过应用本方法，即基于 RNA 提取物的核酸酶 S1 酶消化，然后进行 HPLC 分析，可以定量混合物中的未修饰核苷酸单磷酸 (NMP)，并识别其羟甲基衍生物以及其他非典型 RNA 部分。从一组按照不同方案固定/包埋的小鼠肝脏以及一组年龄在 0 到 30 岁的临床样本中获得的结果表明，基于酒精的固定剂不会在 ISO 标准推荐下诱导核酸的化学修饰，并证实了预分析条件在 RNA 保存中起着重要作用。

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