7700 SEQUENCE DETECTOR AND BIOMEK WORKSTATION

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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.