ABI-PRISM 7700 TO ASSESS MRNA AND DNA

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The ABI-PRISM 7700 is an instrument that uses fluorescence-labeled oligonucleotides to assess mRNA levels by RT-PCR or DNA levels by PCR (termed the Taqman procedure). In other PCR methods, an end-point measurement is employed, but these methods are not as sensitive, accurate or reproducible. With the ABI-Prism, 7700, the PCR cycle number at which the fluorescence signal exceeds a threshold value is used to assess mRNA or DNA levels and consequently, the detection procedure is always in the linear range of PCR. The ABI-Prism 7700 also detects as few as 42 copies or starting material. No post PCR steps are required thereby saving time and expense. Indeed, many of the proposed studies cannot be performed without the novel technology incorporated into the ABI-PRISM 7700 because no other method can assess mRNA or DNA levels from the small amounts of sample material available. A large number of NIH-funded researchers at LSUMC are excited about the benefits of the ABI-Prism 7700, and the opportunities for the collaboration such shared instrumentation represents.. The Major User Group comprises seven individuals from four departments with a wide variety of research interests; this diversity reflects the collaborative nature of LSUMC. Each investigator has extensive experience in PCR and mRNA measurements, and thus has the expertise necessary to utilize the ABI-Prism 7700. The projects by NIH-funded investigators will use the ABI-prism 7700 to: (1) assess cytokine mRNA levels in CD4+ T cells and other selected cell-types in malaria and in Herpes virus infections, (2) quantify organism DNA level for Plasmodium and Herpes virus during infections in mice (3) assess the level of expression of T cell-and lymphoid-specific genes in minor populations of pre-T cells in the bone marrow, (4) assess mRNA levels of ion transport genes in medically important models of the different ion states, (5) assess adhesion molecule expression in tissue and sorted cells from an inflamed bowel, (6) to determine the effects of both cis- and trans-acting mutations on the transcription of HSP90 Genes in yeast; and (7) determine the effects of deletion of equine Herpesvirus genes on transcription of other viral genes. The ABI-Prism 7700 is an expensive, state-of-the-art instrument that will maximize the research efforts of a group of seven NIH-funded researchers. The LSUMC-S administration enthusiastically supports this proposal, and will provide the technical personnel and help cover maintenance costs. The instrument will be part of the Molecular Structure and Dynamics ore Facility; thus the procedures are already in place to maintain, share, monitor and ensure optimal use of this shared instrument.