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研究人员和分析癌症患者组织样本的病理学家可用的分子工具箱。我们的建议详细说明了开发一种新的方法来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。替代方法从提取受损的、质量相对较低的RNA开始，这限制了下游分析。相反，我们的系统利用交联的RNA分子在原位获得可放大的信号，这在分析灵敏度和工作流程简单性方面提供了几个明显的优势。重要的是，我们的系统利用了广泛可用的仪器和试剂，使研究人员和临床医生能够立即获得它。我们在这个项目中寻求实现三个相互重叠的目标：1.开发一个健壮的系统，使用FFPE肿瘤切片在原位产生PCR可扩增信号。这项工作的成功将为研究和临床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测序分析相结合。高通量，或“下一代”DNA测序(NGS)是一项强大的技术，用于分析复杂的DNA序列混合物。我们建议将我们的新方法与NGS分析相结合，以高度多元化地测量FFPE标本中的基因表达。3.空间分辨基因表达的多路测量。该项目的这一部分致力于开发一种新的方法来测量FFPE切片或固定细胞铺展中的大量基因，同时保持原始组织的单细胞分辨率。为此，我们建议采用最近发展的技术来进行“本地”DNA扩增。这样的系统有可能极大地增加研究人员和病理学家通过分析一张显微镜载玻片收集的信息量。

 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.