Ultra Rapid Methods for Streamlined Tissue-to-RT-PCR

描述(由申请人提供)： 这项第二阶段的建议旨在简化、加快和稳定从组织样本中回收高质量的RNA。在第一阶段，我们证明了一种称为“熔融”(多酶组织液化)的不插手、闭管组织破碎法的可行性。Melt利用强大的分解代谢酶在几分钟内液化组织，而不需要侵入性的机械力。熔融后可获得高产量的完整RNA。重要的是，融化的酶破坏细胞核糖核酸酶，并在环境温度下稳定组织裂解物中的RNA长达5天。此外，MELT与新鲜收获的、闪速冷冻的或经RNAIater(R)处理的组织兼容，包括小鼠和人类组织，包括肿瘤标本。综上所述，这些进展有望通过在RNA稳定性、闭管组织破坏和快速单管样品制备方面的创新，为稳定和量化组织中的基因表达提供更快、更简单、更安全和更强大的方法。 在第二阶段，我们将把持续的熔融增强与促进RNA加工的新方法结合起来。首先，我们将加速融化组织的消化，并最大限度地提高所产生的RNA的质量。其次，我们将把熔融改进与新型磁珠联系起来，这种磁珠可以在短短20分钟内提纯不含DNA的RNA。这种方法将确保回收大量的RNA，用于任何表达谱方法的分析，包括微阵列。最后，我们将启用一种专门适用于qRT-PCR(“组织到RT-PCR”)的超快速RNA样本制备策略，它完全跳过了RNA分离。使用组织破坏、核糖核酸酶控制、DNA去除和RT-PCR抑制管理的新方法，我们将在不到10分钟的时间内实现组织到RT-PCR，除了RT-PCR反应本身之外，所有步骤都在同一试管中进行。 成功实现这些目标将产生易于使用的产品，与目前的方法相比，这些产品可以提供更高的RNA产量、更大的样本吞吐量、更容易实现的自动化，以及更低的可变性、污染和生物危害风险。熔融技术的受益者将包括生命科学研究和临床诊断实验室，在这些实验室，新兴的RNA生物标记物可以与更简单的样品制备方法相结合，以加速采用分子诊断程序。

DESCRIPTION (provided by applicant): This Phase II proposal aims to simplify, expedite, and stabilize the recovery of high quality RNA from tissue samples. In Phase I, we demonstrated the feasibility of a hands-off, closed tube tissue disruption method termed "MELT" (Multi-Enzymatic Liquefaction of Tissue). MELT enlists potent catabolic enzymes to liquefy tissue within minutes without invasive mechanical force. High yields of intact RNA are obtained after MELT. Importantly, MELT enzymes destroy cellular RNases and stabilize RNA in tissue lysates for up to 5 days at ambient temperatures. Additionally, MELT is compatible with freshly harvested, flash-frozen, or RNAIater(R)-treated tissues, both mouse and human, and including tumor specimens. Taken together, these advances promise faster, simpler, safer, and more robust methods for stabilizing and quantifying gene expression in tissues through innovations in RNA stability, closed-tube tissue disruption, and rapid single-tube sample preparation. In Phase II we will integrate continuing MELT enhancements with new ways to facilitate RNA processing. First, we will accelerate MELT tissue digestions and maximize the quality of the resulting RNA. Second, we will link MELT improvements with novel magnetic beads that can enable the purification of DNA-free RNA in as little as 20 min. This method will secure the recovery of large amounts of RNA for analysis by any expression profiling method, including microarrays. Last, we will enable an ultra rapid RNA sample preparation strategy specifically suited for qRT-PCR ("Tissue-to-RT-PCR") that skips RNA isolation altogether. Using novel approaches for tissue disruption, RNase control, DNA removal, and the management of RT-PCR inhibition, we will enable Tissue-to-RT-PCR in less than 10 minutes, with all of the steps but the RT-PCR reaction itself occurring in the same tube. Success in these objectives will result in easy-to-use products that offer improved RNA yields, greater sample throughput, more ready automation, and reduced variability, contamination, and biohazard risk compared to current methods. The beneficiaries of MELT technology will include life science researches and clinical diagnostic labs, where emerging RNA biomarkers can be combined with simpler sample preparation methods to hasten the adoption of molecular diagnostics procedures.