The main goal of the Molecular Biology and Biochemistry core is to measure the levels of RNA, protein and or enzyme activities of specific molecules of interest in samples generated by the five different projects. The core will collect samples and process them to isolate the cell-free extracts or the RNA from tissue or cells. The level of messenger RNA will predominantly be examined using state of the art techniques such as quantitative RT-PCR and ribonuclease protection assays. The ABI Prism 7700 sequence detection system and the Taqman approach to real-time PCR allows us to perform RT-PCR in a quantitative manner. As well as quantifying steady-state mRNA levels, we will examine protein levels by western analysis. Many of the molecules of interest also have enzymatic function. We plan to measure the activities of catalase, xanthine dehydrogenase and oxidase, myeloperoxidase, glutathione peroxidase and the different isoforms of superoxide dismutase and nitric oxide synthase using functional biochemical assays. The core also has expertise in electrophoretic mobility shift assays allowing us to study the activity of transcription factors such as NFkB and AP-1. Project 0010 has also proposed studies requiring mammalian cell transfection. The Core Director has extensive experience in this field and the availability of a Biolistic PDS-1000/He Particle Delivery System has allowed the core to perform preliminary studies that demonstrate we can deliver recombinant DNA into human umbilical vein endothelial cells. Centralization of these sections of the projects not only provides techniques that would not otherwise be easily available to each of the Principal Investigators, but also a standardization of the collection of samples, the assay procedure and quantification of results. This allows a comparison of results between the different projects. Dr. Harrison is the Core Director and will supervise Mr. Jeffery Houghton and will be assisted by Drs. Feelisch and Yeh.
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