利用纳米试管技术量化高度降解固定组织中的 mRNA:比较研究

IF 2.3 Q3 BIOCHEMICAL RESEARCH METHODS
E. Azzalini, Barbara Di Stefano, Vincenzo Canzonieri, Tiziana Venesio, Umberto Miglio, C. Marchiò, A. Sapino, C. Previderè, P. Fattorini, Serena Bonin
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引用次数: 0

摘要

档案组织是转化研究中进行分子分析最有用的人体组织来源。这些标本的主要问题是生物大分子(即蛋白质、DNA 和 RNA)的修饰和降解。近十年来,一些高通量分析方法已被应用于档案组织。虽然现在组织学组织是用中性缓冲福尔马林固定的,但在最近的过去,布因溶液也被用于组织处理。本研究旨在探讨 nCounter 纳米环杂交技术与作为 RNA 来源的标准福尔马林固定和石蜡包埋(FFPE)组织相比,量化高度降解样本(如布氏固定和石蜡包埋(BFPE)组织)中 mRNA 的可行性。共分析了 8 位患者的 16 块石蜡包埋组织块(8 块为 FFPE,8 块为 BEPE)。对每个 RNA 样本的 300 纳克应用了纳米环技术,而对 360 纳克的相同模板进行了逆转录并进行了 qPCR 和 ddPCR。结果表明,在检测 FFPE 和 BFPE 样品中的目标 mRNA 方面,Nanostring 技术优于参考方法(ddPCR 和 qPCR)。然而,即使是纳米环技术也无法摆脱 RNA 模板降解的限制,这可能会导致对基因表达水平得出误导性结论。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study
Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin’s solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin’s fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.
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来源期刊
Methods and Protocols
Methods and Protocols Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (miscellaneous)
CiteScore
3.60
自引率
0.00%
发文量
85
审稿时长
8 weeks
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