NOVEL RNASE PROTECTION ASSAY FOR CYTOKINE MRNAS

描述(改编自申请人的摘要)：测量 重要的信使核糖核酸已成为研究其影响的关键 药物、生物效应物和生长条件。细胞因子发挥着特别的作用 在生长控制和生物反应中发挥重要作用。但是，当前 化验通常使用放射性标签，在易用性方面受到限制。这个 研究人员建议构建一种廉价的大阵列检测系统，用于 以细胞因子mRNAs为模型靶点的基于核糖核酸酶保护的RNA。化验结果 利用融合了RNase不稳定链和酶的嵌合探针 附属于5‘端的记者团。在没有靶RNA的情况下， 报告基团从探针上裂开，从而防止在适当的 衍生多井板。在靶mRNA杂交体存在的情况下， 不稳定的连锁不受核糖核酸酶的影响。处理后剩余的酶的量 核糖核酸酶的裂解与杂交后的mRNA成正比。 第一阶段旨在确定使用该系统的可行性 提高小鼠白细胞介素7(IL-7)的易用性和检测灵敏度 以m RNA为模型。主要技术创新有：1)施工 酶标记的嵌合捕获探针，以及2)一种 通用捕捉板宏阵列，检测受保护的碎片。同相 我们打算将这些产品中的第一个引入到研究中 市场。 建议的商业应用： 建议的商业应用是用于测量信使核糖核酸种类的检测试剂盒。 (例如，白介素7)在从基础研究到药物的各种应用中 发现号。目标市场包括通过制药的基础研究实验室 公司。

DESCRIPTION (Adapted from applicant's abstract): Measuring the levels of important mRNA species has become essential in investigating the effect of drugs, biological effectors and growth conditions. Cytokines play an especially important role in growth control and biological response. However, current assays typically employ radioactive label and are limited in ease of use. The investigators propose to construct an inexpensive macroarray assay system for RNA based on RNase protection using cytokine mRNAs as a model target. The assay utilizes chimeric probes incorporating an RNase labile linkage and an enzymatic reporter group attached to the 5' terminus. In the absence of target RNA, the reporter group is cleaved from the probe thus preventing capture on a suitably derivatized multiwell plate. In the presence of the target mRNA hybrid, the labile linkage is protected from Rnase. The amount of enzyme remaining after Rnase cleavage is directly proportional to the mRNA hybridized. Phase I is designed to determine the feasibility of using this system to improve ease of use and detection sensitivity for mouse interleukin 7 (IL-7) mRNA as a model. The principal technical innovations are: 1) the construction of the enzyme labeled chimeric capture probe, and 2) the development of a universal capture plate macroarray to detect the protected fragments. In Phase III we intend to introduce the first of these products into the research market. PROPOSED COMMERCIAL APPLICATION: Proposed commercial applications are assay kits for the measurement of mRNA species (such as interleukin 7, for example) in applications ranging from basic research to drug discovery. Target markets include basic research laboratories through pharmaceutical companies.