Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking
Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking
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DOI：
10.1089/bio.2021.0023
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发表时间：
2021-09-29
影响因子：
1.6
通讯作者：
Rimsza, Lisa
中科院分区：
文献类型：
--
作者：
Ramsower, Colleen;Wisner, Lee;Rimsza, Lisa
背景：福尔马林固定石蜡包埋（FFPE）组织是临床和基础科学研究的宝贵资源。石蜡块和由此产生的未染色切片 (USS) 在使用前通常会保存数年。之前的研究评估了时间、温度、湿度和惰性气体对USS保存的影响；然而，没有研究同时检查所有四个变量。 方法：在目前的工作中，我们前瞻性地、盲目地评估了 0 至 24 个月的时间点、室温与冷藏温度，以及来自北美和非洲的各种良性和恶性组织中干燥剂和/或氮气气氛的存在。终点包括免疫组织化学 (IHC)、原位杂交 (ISH)、提取的 RNA 和 DNA 的数量和质量，以及管家基因和肿瘤特异性基因的新型多重数字基因表达谱分析中的信使 RNA 性能。结果：我们发现，使用当前的抗原修复、染色和提取方法，IHC、ISH、RNA 和 DNA 的终点在各种测试条件下都得到了很好的保存，这表明预嵌入因素会导致后续组织的变异性诚信。我们还记录了分光光度法对核酸浓度的估计通常高于荧光测定法，这可能与最终测定的开发有关。我们进一步描述了一种新的多重检测，即 PlexSet 数字基因表达检测，适用于评估 FFPE 组织中的 RNA 质量。结论：总之，这些结果可能为 USS 长期储存条件的方法提供有用的指导。
Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together.Methods: In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC), in situ hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes.Results: We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues.Conclusion: Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS.