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

描述（由申请人提供）： 该第二阶段提案旨在简化、加快和稳定从组织样品中回收高质量RNA。在第一阶段，我们证明了一种称为“MELT”（组织的多酶液化）的免干预封闭管组织破碎方法的可行性。MELT利用强效分解代谢酶在几分钟内使组织分解，而无需侵入性机械力。在MELT之后获得高产量的完整RNA。重要的是，MELT酶破坏细胞RNA酶，并在环境温度下使组织裂解物中的RNA稳定长达5天。此外，MELT与新鲜收获的、快速冷冻的或RNAIater（R）处理的组织相容，包括小鼠和人的肿瘤标本。总的来说，这些进展有望通过RNA稳定性、闭管组织破坏和快速单管样品制备方面的创新，实现更快、更简单、更安全和更稳健的方法来稳定和定量组织中的基因表达。 在第二阶段，我们将整合持续的MELT增强与新的方法，以促进RNA加工。首先，我们将加速MELT组织的消化，并最大限度地提高所得RNA的质量。其次，我们将MELT的改进与新型磁珠相结合，这种磁珠可以在短短20分钟内纯化无DNA的RNA。这种方法将确保回收大量RNA，用于任何表达谱分析方法，包括微阵列。最后，我们将实现一种特别适用于qRT-PCR（“组织-RT-PCR”）的超快速RNA样品制备策略，该策略完全跳过RNA分离。使用新的方法进行组织破坏，RNA酶控制，DNA去除和RT-PCR抑制的管理，我们将在不到10分钟的时间内实现组织到RT-PCR，除了RT-PCR反应本身之外的所有步骤都发生在同一个试管中。 这些目标的成功将导致易于使用的产品，与现有方法相比，这些产品提供更高的RNA产量，更大的样品通量，更容易自动化，并降低变异性，污染和生物危害风险。MELT技术的受益者将包括生命科学研究和临床诊断实验室，新兴的RNA生物标志物可以与更简单的样本制备方法相结合，以加速分子诊断程序的采用。

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.