CORE--MOLECULAR ANALYSIS

核心--分子分析

• 批准号: 6324544
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6324544
• 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人员提供分子分析服务。蛋白质免疫印迹、Northern 杂交和原位杂交以及[定量、竞争]聚合酶链反应 (RT-PCR) 分析； [DNA片段化；电动势变化测定；乙酰胆碱 (ACh) 相关酶和 ICE 酶测定；和细胞因子周期蛋白的细胞因子生物活性分析]现已可用。此外，还可根据要求提供其他可能感兴趣的蛋白质及其编码 mRNA（例如其他细胞因子、生长因子或结构蛋白）的分析。可以使用 S100beta 和 GFAP ELISA，并且可以构建其他 ELISA 来快速筛选大量样品。作为细胞培养物中特定 mRNA 的快速筛选，还可以使用微原位杂交。 [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）。] 在此核心中进行的分析的数据将作为原始数据和初步分析数据提供给项目研究人员。例如，来自 Northern 和 Western 免疫印迹分析和 RT-PCR 的数据将作为原始数据（凝胶图片和转移胶片）和相对于总蛋白、总 RNA、G3PDH 或可杂交核糖体 RNA 的 ng/μl 提取物返回给研究者。 ELISA 数据将以每个样品的光密度单位的形式作为原始数据返回，同时生成标准曲线，并计算 ng/mug 蛋白质和 ng/mul 提取物。原位杂交后，切片或细胞培养物将返回给研究者，或者，如果需要，将指示发送给[项目 4] 进行图像分析。来自培养物微原位杂交的数据将以相对于总聚腺苷酸化mRNA的cpm/孔的形式返回给研究者。 [对该核心中执行的服务的验证和价值将存档在该核心的独立计算机数据库中。]