Validation of Vacuum-Based Refrigerated System for Biobanking Tissue Preservation: Analysis of Cellular Morphology, Protein Stability, and RNA Quality
Validation of Vacuum-Based Refrigerated System for Biobanking Tissue Preservation: Analysis of Cellular Morphology, Protein Stability, and RNA Quality
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DOI：
10.1089/bio.2013.0065
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发表时间：
2014-02-01
影响因子：
1.6
通讯作者：
Landriscina, Matteo
中科院分区：
文献类型：
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作者：
Condelli, Valentina;Lettini, Giacomo;Landriscina, Matteo
新鲜、未固定的人类正常和恶性组织的生物库是转化癌症研究和分子病理学中基因表达分析的宝贵来源。然而，分子和细胞分析在临床和转化研究中的成功很大程度上取决于新鲜人体组织样本的收集、处理、储存和质量控制。本研究的目的是评估一种创新的真空制冷系统，将其作为一种逻辑上可行的技术来增加组织标本的收集，保持细胞和分子成分的完整性。我们使用对研究和诊断重要的终点（包括组织形态、表位稳定性和 RNA 完整性）测试了随机选择的 4°C 真空下储存的组织。通过对选定的管家基因和组织特异性基因进行定性和定量 RT 分析来评估基因表达。组织形态和总体蛋白质稳定性通常保存完好，仅在胆囊组织中受到损害。相比之下，磷蛋白和 RNA 分析表明降解程度随时间变化，从 24 小时到 72 小时逐渐丧失稳定性。然而，RNA 质量的降低并不代表对选定基因的成功表达分析的限制。事实上，比较定性和定量 RT-PCR 分析表明，从真空储存的组织中提取的 RNA 适用于基因表达谱分析，但需要高度灵敏的技术，例如定量 RT-PCR。这些数据表明，基于冷冻真空的系统代表了一种适合且可行的技术，用于将新鲜标本从手术室常规运输到生物库，从而增加了收集生物标本的机会。
Biobanks of fresh, unfixed human normal and malignant tissues represent a valuable source for gene expression analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis in both clinical and translational research is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. The aim of this study was to evaluate an innovative vacuum-based refrigerated system, as a logistically feasible technology to increase the collection of tissue specimens, preserving the integrity of cellular and molecular components. We tested randomly-selected tissues stored under vacuum at 4°C by using endpoints important for research and diagnosis, including tissue morphology, epitope stability, and RNA integrity. Gene expression was evaluated by qualitative and quantitative RT analysis of selected housekeeping and tissue-specific genes. Tissue morphology and overall protein stability were generally well preserved, being compromised only in gallbladder tissue. By contrast, phosphoprotein and RNA analysis demonstrated a time-dependent degree of degradation, with progressive loss of stability from 24 to 72 hours. However, this reduction in RNA quality did not represent a limitation for successful expression analysis of selected genes. Indeed, a comparative qualitative and quantitative RT-PCR analysis showed that RNA extracted from tissues stored under vacuum is suitable for gene expression profiling, but requires highly sensitive technologies, such as quantitative RT-PCR. These data suggest that the refrigerated vacuum-based system represents a suitable and feasible technology for routine transport of fresh specimens from surgery to biobanks, thus increasing the opportunity to collect biospecimens.