Validation of Gene Expression Based Toxicity Screening

描述(由申请人提供)： 这项建议的总体目标是提供一条途径，将基于微阵列的癌症和毒理学研究转化为成本效益高、高通量、有效的分析方法，用于评估一系列不同的毒理学和肿瘤学机制终点。这一建议的核心是使用我们在实验室开发的高度多元化、定量的、基于RTPCR的基因表达分析方法。表达谱(XP)方法结合了在PCR扩增过程中使用基因特异性和通用引物来锁定基因比率的方法，通常用于扩增每个反应包含20-30个基因的多重体。该方法的优点包括：(A)通过使用标准的现成试剂和设备，每次检测成本低，(B)每天高通量1000个样品，自动化更多，以及(C)高灵敏度，每次反应仅使用5 ng总RNA即可检测每个细胞的单拷贝转录本。现成试剂和设备的使用也意味着该分析可以在一系列学术、企业和临床环境中轻松进行。 我们小组建议识别和验证一组可用于新化疗和化学预防药物的详细毒理学评估的基因。在这项拟议工作的第一阶段，我们打算重点开发和验证两个特定毒理学终点的基因集，遗传毒性和非遗传毒性致癌性。这一过程将涉及对原代大鼠肝细胞和一组约80种具有已知遗传毒性和非遗传毒性活性的化合物进行剂量反应研究。XP方法将被用来监测大约100个已知与毒素反应有关的基因的表达水平。对这些基因数据的分析将被用来识别预测遗传毒性和非遗传毒性活动的较小一组基因。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to provide a pathway for translating microarray-based research in cancer and toxicology into cost efficient, high throughput, validated assays for the assessment of a range of different toxicological and oncological mechanistic endpoints. Central to this proposal is the use of a highly multiplexed, quantitative, rtPCR-based gene expression analysis method that we have developed in our lab. The expression profiling (XP) methodology incorporates the use of gene-specific and universal primers to lock gene ratios during PCR amplification and is generally used to amplify multiplexes containing 20-30 genes per reaction. Advantages of the method include (a) low cost per assay via the use of standard, off-the-shelf reagents and equipment, (b) high throughput of >1000 samples per day, more with automation, and (c) high sensitivity, detecting single copy per cell transcripts using as little as 5 ng of total RNA per reaction. The use of off-the-shelf reagents and equipment also means that the assay can be readily performed in a range of academic, corporate and clinical settings. Our group proposes to identify and validate a set of genes that can be used for detailed toxicological evaluation of new chemotherapy and chemopreventive agents. In the Phase I of this proposed work, we intend to focus on the development and validation of gene sets for two specific toxicological endpoints, genotoxic and non-genotoxic carcinogenicity. The process will involve the performance of dose response studies with primary rat hepatocytes and a set of approximately 80 compounds with known genotoxic and non-genotoxic activities. The XP method will be used to monitor the expression levels of approximately 100 genes that are known to be involved in response to toxins. Analysis of these gene data will be used to identify a smaller set of genes that are predictive of genotoxic and non-genotoxic activities.