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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：
9530754
负责人：
Harry Benjamin Larman
金额：
$ 8.18万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9530754
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.