7700 SEQUENCE DETECTOR AND BIOMEK WORKSTATION

目前激素和药物作用的细胞和分子基础研究的一个主要限制是缺乏对生物系统中特定基因转录物进行精确和可靠定量的方便技术。目前可用于测量特定mRNA的技术要么非常不敏感且不精确（Northern印迹），要么需要多种复杂的实验操作（RNA酶保护或竞争性Q-PCR）。“实时”定量RT-PCR通过促进在“真实的时间”中直接测量PCR扩增的进展而提供了对该问题的新颖解决方案。通过在PCR扩增的指数阶段收集数据，并通过消除对电泳和单个扩增反应的定量的需要，该系统允许对特定mRNA进行精确、可重复和简单的定量。ABI最近开发了一种能够支持96孔阵列格式的“实时”PCR反应的多路复用仪器，允许对多种转录物进行高通量分析。当与贝克曼机器人工作站相结合时，该仪器可用于在一天内生成大量转录本的定量数据。我们建议建立一个核心Q-PCR设施，由这些仪器组成，在熟练的研究科学家的监督下，参与研究的研究人员可以测量特定研究项目感兴趣的特定转录本。这样一个设施的可用性不仅将扩大目前资助的研究项目的范围，但将提供机会，开发新的实验方法，以前不可行的，因为传统的分析程序的后勤限制。

A major limitation in current investigations of the cellular and molecular basis of hormone and drug action is the lack of convenient techniques for the precise and reliable quantitation of specific gene transcripts in biological systems. The current techniques available for the measurement of specific mRNA's are either very insensitive and imprecise (Norther blots_ or require multiple complex experimental manipulations (RNAase protection or competitive Q-PCR). "Real-time" Quantitative RT-PCR offers a novel solution to this problem by facilitating the direct measurement of the progress of a PCR amplification in "real time". By gathering data during the exponential phase of the PCR amplification and by eliminating the need for electrophoresis and quantitation of individual amplification reactions, this system allows for the precise, reproducible and simple quantitation of specific mRNAs. ABI has recently developed a multiplexed instrument capable of supporting "real-time" PCR reactions in a 96-well array format, allowing for the high throughput analysis of multiple transcripts. When coupled with a Beckman robotic workstation, this instrument can be used to generate quantitative data on large numbers of transcripts in a single day. We propose to establish a core Q-PCR facility comprised of these instruments under the supervision of a skilled research scientists that will be available to participating investigators for the measurement of specific transcripts of interest to particular research projects. The availability of such a facility will not only extend the scope of current funded research projects but will provide the opportunity to develop novel experimental approaches not previously feasible because of the logistical limitations of conventional assay procedures.