CORE--MOLECULAR ANALYSIS

核心--分子分析

• 批准号: 6098595
• 负责人: Sue Tilton Griffin
• 金额: $ 15.3万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6098595
• 关键词: acetylcholinesterase; analytical method; animal tissue; bioassay; biomedical facility; choline acetyltransferase; cytokine; enzyme activity; enzyme linked immunosorbent assay; gel mobility shift assay; human genetic material tag; human tissue; in situ hybridization; messenger RNA; molecular biology; northern blottings; polymerase chain reaction; tissue /cell culture; western blottings

The purpose of Core C is to provide molecular analysis services to the Project investigators in this Program Project. Western immunoblot, northern and in situ hybridization, and [quantitative, competitive] polymerase chain reaction (RT-PCR) analyses; [DNA fragmentation; electromotive shift assay; enzyme assays for acetylcholine (ACh)-related enzymes and for ICE; and cytokine bioactivity analysis for cytokine cycle proteins] are now available. In addition, analysis of other proteins and their encoding mRNAs that may be of interest (e.g., other cytokines, growth factors, or structural proteins) will be available upon request. S100beta and GFAP ELISAs are available, and other ELISAs can be constructed for rapid screening of large numbers of samples. As a rapid screen for specific mRNAs in cell cultures, micro-in situ hybridization is also available. [The tissue to be analyzed will from human and rodent brain (Projects 1-4), and from cell cultures (Project 3).] Data from analyses performed in this Core will be provided to the Project Investigators as both raw data and preliminary analyzed data. For example, data from northern and western immunoblot analyses and RT- PCR will be returned to the investigator as raw data (pictures of gels and films of transfers) and as ng/mul of extract, relative to total protein, total RNA, G3PDH, or hybridizable ribosomal RNA. Data from ELISAs will be returned as raw data in the form of optical density units per sample, together with a standard curve generated at the same time, and calculation of ng/mug protein and ng/mul extract. Following in situ hybridization, sections or cell cultures will be returned to the investigator or, if requested, sent direction to [project 4] for image analysis. Data from micro-in situ hybridization of cultures will be returned to the investigator as cpm/well relative to total polyadenylated mRNA. [Verification of and values from services performed in this Core will be archived in the free standing computer database in this core.]

核心C的目的是为本计划项目的项目研究者提供分子分析服务。现在可以使用蛋白质免疫印迹、北方和原位杂交以及[定量、竞争性]聚合酶链反应（RT-PCR）分析; [DNA片段化;电动位移分析;乙酰胆碱（ACh）相关酶和ICE的酶分析;以及细胞因子周期蛋白的细胞因子生物活性分析]。此外，对可能感兴趣的其他蛋白质及其编码mRNA的分析（例如，其它细胞因子、生长因子或结构蛋白）将根据要求提供。S100 β和GFAP ELISA是可用的，并且可以构建其他ELISA用于快速筛选大量样品。作为在细胞培养物中快速筛选特异性mRNA的方法，也可使用微量原位杂交。[The待分析的组织将来自人类和啮齿动物的大脑（项目1-4），以及细胞培养物（项目3）。]将向项目研究者提供在本核心中进行的分析数据，作为原始数据和初步分析数据。例如，来自北方和Western免疫印迹分析和RT-PCR的数据将作为原始数据（凝胶照片和转移膜）和相对于总蛋白、总RNA、G3 PDH或可杂交核糖体RNA的ng/穆尔提取物返回给研究者。ELISA的数据将作为原始数据以每份样本的光密度单位的形式返回，同时生成标准曲线，并计算ng/mug蛋白质和ng/穆尔提取物。原位杂交后，将切片或细胞培养物返还给研究者，或如有要求，直接发送至[项目4]进行图像分析。培养物的微量原位杂交数据将以相对于总聚腺苷酸化mRNA的cpm/孔返回给研究者。[在本核心中执行的服务的验证和价值将存档在本核心的独立计算机数据库中。]