Many of the specific aims in projects 1-4 require quantification of mRNA and/or protein levels in treated samples. While for practical purposes initial extraction of protein or RNA will be performed on site for each project, the Molecular core will undertake routine RT-PCR quantification and Western analysis as well as aid in the preparation of plasmids and probes for use in specific RPA assays. This will be undertaken as a core because of the need for experience in execution for the RT-PCR assays, as well as to improve/ensure consistency of analysis between labs in the case of Western analysis and RT-PCR assay. The Core will also undertake a major role in development of new mRNA quantification assays either through the design and initial optimization of both RT-PCR and RPA assays, including isolation of previously unavailable target sequences from ovine or human RNA or cDNA libraries (ovine and human endothel8ial cell or trophoblast), or alternatively in the use of newer cDNA array methodology recently established in the PI's laboratory. Similarly, the core will undertake a major role in development of western analysis procedures using new anti-sera, with blocking buffer optimization, as well as first and second antibody optimization. The PI, CoI and named Technician listed all have considerable experience in each of these areas. In addition to these specific assays, the core will act as a coordination and distribution point for bulk purchased molecular reagents (so reducing unit cost), and labile supplies and a storage/coordination facility for precious resources (plasmids, libraries, and standards as well as antisera and protein standards). The staff will also assist the participants in projects 1- 4 with primer design and sequence analysis as required.
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