The objective of this proposal is to seek support for the purchase of a real-time PCR machine (PE 7700 Sequence Detector). The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. As little as a single copy of a particular sequence can be specifically amplified and detected. Theoretically, there is a quantitative relationship between amount of starting target sequence and amount of PCR product at any given cycle. In practice, though, it is a common experience for replicate reactions to yield different amounts of PCR product. The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the routine and reliable quantification of PCR products. J.A. Olschowka (principal investigator) and M.K. O'Banion (major user) routinely use competitive quantitative RT-PCR in their NIH-funded work examining inflammation in the central nervous system. Other major users, all of whom are funded by NIH, propose to use the PE 7700 for quantifying DNA and/or RNA in a variety of settings. D.A. Figlewicz will use the instrument in expression and mapping studies of candidate genes potentially involved in Facioscapulohumeral Dystrophy. J. Finkelstein studies cytokine and chemokine expression in acute lung injury and progressive pulmonary fibrosis. Currently using RNAse protection assays (RPA) to analyze mRNA levels, his work will be accelerated by real-time PCR. Dr. Rodier will use the instrument in her molecular studies of autism. The sensitivity of the instrument will be especially important to her when working with small amounts of mouse embryo tissue. Dr. Phipps studies the role of CD 40 in lung injury as well as the regulation of immunity by prostaglandins. Again, analysis of mRNA levels in these studies will be enhanced over current RPA methods in terms of speed, reliability and reproducibility. The instrument will be housed in the Molecular Biology core facility within the Department of Neurobiology and Anatomy. This core facility is the responsibility of the J. A. Olschowka (the Principal Investigator) who will oversee the daily use of the instrument. A committee made up of the Major Users will provide long-term guidance about the use of the instrument. The institution will provide a room of approximately 350 sq feet to house the instrument, ancillary equipment required for PCR, and cost-sharing of service contracts.
