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Expression profiling from microdissected samples

显微解剖样本的表达谱

基本信息

批准号：
6731128
负责人：
MARIANNA Mansfield GOLDRICK
金额：
$ 37.15万
依托单位：
AMBION, INC.
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-01 至 2006-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6731128
关键词：
RNA clinical research cytogenetics expression cloning flow cytometry fluorescent dye /probe green fluorescent proteins human tissue laboratory mouse laser capture microdissection lasers method development microarray technology northern blottings nucleic acid purification nucleic acid quantitation /detection tissue /cell culture tumor antigens

项目摘要

DESCRIPTION (provided by applicant): One objective of the proposal is to develop a product line that will consist of amplified RNA (aRNA) derived from pure populations of cells from various regions of mouse brain. The target cells will be selected by Laser Capture Micro-dissection (LCM). The product line will include aRNA derived from normal mice and from mutant mice that serve as models of human neurodegenative disorders, including Alzheimer's disease, Huntington's disease, and ataxia telangiectasia. The product will be targeted to researchers carrying out expression profiling studies that aim to understand the molecular basis of normal neurological function and the molecular pathology of neurological disease. During the first year, efforts will be focused on scaling up procedures for sample processing, micro-dissection, and RNA isolation and amplification from normal mice. During the second year the procedures will be applied to the mutant strains. The aRNA will be produced using T7-mediated in vitro transcription. In order to help meet the anticipated Production-scale goals, efforts will be directed to adapting the procedures, especially RNA amplification, to a robotic platform. In order to assess the RNA from micro-dissected samples for quality assurance purposes, molecular markers will be identified to use for verifying that the RNA is derived from the intended cell population. Efforts will also be directed to improving methods for identifying target cells for micro-dissection in samples such as tumors, where target cells (e.g. malignant cells) cannot always be distinguished by histological staining. The methods will be compatible with isolation of intact RNA from the selected cells, so that it can be amplified for use in microarray expression profiling assays. To meet this goal we will develop rapid fluorescent detection methods using primary fluorescent antibodies directed to tumor antigens, and we will also adapt detection of target cells in transgenic animals expressing Green Fluorescent Protein for use with LCM. Achieving these goals will provide aRNA from distinct cellular subtypes to neuroscience researchers who do not currently have access to this resource. Use of aRNA from defined cell subtypes, as opposed to bulk tissue, will improve the ability to make biologically meaningful conclusions from expression profiling experiments carried out on highly heterogeneous tissues such as brain. Another goal of the project is to release a kit for recovery of high-quality total RNA from microdissected samples, especially those obtained by LCM. The kit will be of general use to life science researchers working with micro-scale samples.

描述（由申请人提供）：该提案的一个目标是开发一条产品线，该产品线将由来自小鼠大脑不同区域的纯细胞群的扩增RNA（aRNA）组成。将通过激光捕获显微切割（LCM）选择靶细胞。该产品线将包括来自正常小鼠和突变小鼠的aRNA，突变小鼠作为人类神经退行性疾病的模型，包括阿尔茨海默病，亨廷顿病和共济失调毛细血管扩张症。该产品将针对进行表达谱研究的研究人员，旨在了解正常神经功能的分子基础和神经疾病的分子病理学。在第一年，工作重点将放在扩大样本处理，显微解剖和RNA分离和扩增正常小鼠的程序。在第二年，将对突变菌株应用该程序。将使用T7介导的体外转录产生aRNA。为了帮助实现预期的生产规模目标，将努力使程序，特别是RNA扩增，适应机器人平台。为了评估来自显微切割样本的RNA以用于质量保证目的，将鉴定分子标记物以用于验证RNA是否来自预期细胞群。还将努力改进用于鉴定样品如肿瘤中用于显微切割的靶细胞的方法，其中靶细胞（例如恶性细胞）不能总是通过组织学染色区分。所述方法将与从所选细胞分离完整RNA相容，使得其可以扩增用于扩增。用于微阵列表达谱分析。为了实现这一目标，我们将开发使用针对肿瘤抗原的一级荧光抗体的快速荧光检测方法，并且我们还将调整表达绿色荧光蛋白的转基因动物中的靶细胞的检测以用于LCM。实现这些目标将为目前无法获得此资源的神经科学研究人员提供来自不同细胞亚型的aRNA。与大量组织相反，使用来自确定的细胞亚型的aRNA将提高从在高度异质性组织如脑上进行的表达谱分析实验中得出生物学上有意义的结论的能力。该项目的另一个目标是发布一个试剂盒，用于从显微切割样品中回收高质量的总RNA，特别是通过LCM获得的样品。该工具包将普遍用于生命科学研究人员与微型样品的工作。