A Highly Multiplexed Gene Expression Platform for Fixed Tissue Specimens

用于固定组织样本的高度多重基因表达平台

基本信息

批准号：
9251791
负责人：
Harry Benjamin Larman
金额：
$ 20.25万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9251791
关键词：
Adopted Archives Biological Assay Biopsy Cancer Patient Cell Line Cells Chemicals Clinical Communities Complementary DNA Complex Complex Mixtures DNA DNA Microarray Chip DNA Sequence DNA amplification DNA sequencing Data Set Development Excision Formalin Foundations Gene Expression Gene Expression Profiling Genes Genotype Glean Goals High-Throughput DNA Sequencing Housekeeping Gene Human Cell Line Hybrids Image Immune system In Situ In Situ Hybridization Ligation Literature Measurement Measures Messenger RNA Methodology Methods Microscope Molecular Noise Oligonucleotides Paraffin Paraffin Embedding Pathologist Patient Care Patients Performance Phenotype Population Process Protocols documentation RNA RNA Ligase (ATP)RNA Sequences RNA analysis Reagent Research Research Personnel Resected Resolution Reverse Transcription Sampling Sequencing By Hybridizations Signal Transduction Slide Spatial Distribution Specificity Specimen Surface Suspensions System Techniques Technology Therapeutic Intervention Time Tissue Embedding Tissues Transcript Tumor Tissue Tumor-Derived Waxes accurate diagnosis anticancer research base cost crosslink density experience high dimensionality improved instrumentation molecular pathology next generation novel precision medicine public health relevance sample collection sample fixation success tumor tumor xenograft

项目摘要

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.

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同时测量表达的新方法的开发。福尔马林固定石蜡包埋 (FFPE) 组织切片中存在大量基因。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。相比之下，我们的系统利用交联的 RNA 分子来获取。可原位放大信号，这提供了与检测灵敏度和工作流程简单性相关的几个独特优势，重要的是，我们的系统利用广泛可用的仪器和试剂，使研究人员和牧师可以立即使用它，我们力求在该项目中实现三个重叠的目标：1。开发了一个使用 FFPE 肿瘤切片原位产生 PCR 扩增信号的强大系统，这项工作的成功将为研究和临床界提供一种对 FFPE 标本和固定悬浮细胞进行基因表达分析的全新方法。与高通量 DNA 测序分析集成高通量或“下一代”DNA 测序 (NGS) 是一种用于分析复杂 DNA 序列混合物的强大技术，我们建议将我们的新方法与 NGS 分析集成，以实现基因的高度多重测量。 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来测量 FFPE 切片或固定细胞扩散中的大量基因，同时保留单个基因。为此，我们建议采用最近开发的“局部”DNA 扩增技术，这样的系统有可能显着增加研究人员和病理学家通过分析单个DNA 能够收集到的信息量。显微镜载玻片。