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Abstract：
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.
摘要：
存档组织是可用于转化研究中分子分析的人体组织的最可用来源。这些标本的主要问题是生物分子的修饰和降解，即蛋白质，DNA,RNA。在过去的十年里，几种高通量分析方法已应用于存档组织。尽管现在组织学组织被固定在中性缓冲的福尔马林中，在最近的过去，Bouin's溶液也用于组织处理。本研究旨在探讨nCounterNanostring杂交在高度降解样品中定量mRNA的可行性。如Bouin的固定和石蜡包埋(BFPE)组织，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。分析了来自8名患者的总共16个石蜡包埋的组织块(8个为FFPE,8个为BEPE)。Nanostring技术被应用于300ng的每个RNA样品，而360ng相同的模板被逆转录并提交qPCR和ddPCR。我们的结果表明，Nanostring技术在检测FFPE和BFPE样品中的靶mRNA方面优于参考方法（ddPCR和qPCR）。然而,即使是Nanostring技术也不能逃脱RNA模板降解的限制，这可能会导致基因表达水平上的误导性结论。
