A Highly Multiplexed Gene Expression Platform for Fixed Tissue Specimens

 描述（由申请人提供）：本申请中提出的技术有可能极大地扩展分析癌症患者组织标本的研究人员和病理学家可用的分子工具箱。我们的提案详细介绍了一种新的方法，同时测量福尔马林固定和石蜡包埋（FFPE）组织切片中大量基因的表达的发展。替代方法开始提取受损的、相对低质量的RNA，这限制了下游分析。相比之下，我们的系统利用交联的RNA分子原位获得可重复的信号，这提供了与测定灵敏度和工作流程简单性相关的几个明显优势。重要的是，我们的系统利用广泛可用的仪器和试剂，使研究人员和临床医生可以立即使用。我们在这个项目中寻求实现三个重叠的目标：1。开发一种使用FFPE肿瘤切片原位产生PCR可扩增信号的稳健系统。这一努力的成功将为研究和临床社区提供一种全新的方法来进行FFPE标本和固定悬浮细胞的基因表达分析。2.与高通量DNA测序分析集成。高通量或“下一代”DNA测序（NGS）是用于分析DNA序列的复杂混合物的强大技术。我们建议将我们的新方法与NGS分析相结合，用于FFPE标本中基因表达的高度多重测量。3.空间分辨基因表达的多重测量。该项目的这一部分致力于开发一种新的方法，用于测量FFPE切片或固定细胞涂片中的大量基因，同时保留原始组织的单细胞分辨率。为此，我们建议采用最近开发的“本地”DNA扩增技术。这样的系统有可能大大增加研究人员和病理学家通过分析单个显微镜载玻片所能收集的信息量。

 DESCRIPTION (provided by applicant): The technologies presented in this application have the potential to greatly expand the molecular toolbox available to the researchers and pathologists who analyze cancer patient tissue specimens. Our proposal details the development of a new methodology to simultaneously measure the expression of a large number of genes in formalin fixed and paraffin embedded (FFPE) tissue sections. Alternative approaches begin with extraction of damaged, relatively low quality RNA, which limits downstream analysis. In contrast, our system takes advantage of the crosslinked RNA molecules to obtain amplifiable signals in situ, which provides several distinct advantages related to assay sensitivity and workflow simplicity. Importantly, our system utilizes widely available instrumentation and reagents, making it immediately accessible to researchers and clinicians. We seek to achieve three overlapping goals in this project: 1. Development of a robust system to generate PCR amplifiable signals in situ using FFPE tumor sections. The success of this effort will provide the research and clinical community with a completely new method of performing gene expression analysis of FFPE specimens and fixed suspension cells. 2. Integration with high throughput DNA sequencing analysis. High throughput, or "next generation" DNA sequencing (NGS) is a powerful technology for analyzing complex mixtures of DNA sequences. We propose to integrate our new methodology with NGS analysis for the highly multiplexed measurement of gene expression in FFPE specimens. 3. Multiplexed measurement of spatially resolved gene expression. This part of the project is devoted to the development of a novel methodology for measuring a large number of genes in FFPE sections or fixed cell spreads, while retaining single cell resolution of the original tissue. To this end, e propose to adapt recently developed techniques for 'local' DNA amplification. Such a system has the potential to dramatically increase the amount of information that researchers and pathologists are able to glean by analyzing a single microscope slide.