Director: Iordanis Karagiannidis, PhD
Co-Director: Charalabos Pothoulakis, MD
The main objective of this core is to provide high quality RNA, DNA and protein isolation services to Projects 1 and 2 of the UCLA SCOR for Real time PCR, ELISA or multiplex immunoassays and DNA methylation studies. Additional services include consultation for generation of transgenic animals, over-expression of silencing shRNAs, and for design and testing of the appropriate vectors for the creation of the aforementioned models. The Core is also responsible for storing all of the human blood and tissue samples generated by UCLA SCOR studies. The existence of the core provides standardization for the analysis of samples from several studies which may produce more consistent data across the groups serviced.
The Core has acquired several state-of-the art equipment in order to perform the analysis required for UCLA SCOR projects. In particular the Core contains a Fast Real-time PCR machine for high volume mRNA expression analysis, micro plate readers for protein detection via ELISA and is acquiring a multiplex array reading system (Bio-Plex 3D) for high throughput immunoassay as well as mRNA expression analysis. In addition to its multiplexing capabilities, the Bio-Plex 3D will allow us to consolidate our samples (384-well plate reading capacity) within fewer runs, thus increasing the consistency and reproducibility of our data. Furthermore the core includes an EchoMRI 4-in-1 for (what is considered) the most accurate evaluation of small animal (mouse and rat) body and tissue composition. This equipment has been routinely used by our and other groups that participate in this renewal application. There is also significant expertise within the core for isolation of high quality nucleic acids and protein for analysis for the first two projects of this application.
The added responsibilities of the Core in the current SCOR renewal period stem from the addition of measurements of adipokine expression in blood and adipose tissue (included in Projects 1 and 2) identifying this tissue as a potential target and/or contributor to the effects of stress. In addition, one of the novel contributions of the core includes consultation and participation in the construction of adipose-specific constructs to be used in project 2. Overall, and although the actual construct preparation will be done at the UCLA vector core facilities, our core’s adipose-specific expertise as well as adipose-associated cell culture tools used for vector efficiency testing before introduction to the animals, will be central for the successful application of these unique tools. Finally, the acquisition of high throughput equipment with multiplexing capabilities for both immunoassays (mainly ACTH and cortisol) and probe hybridization-based RNA analyses (GR in both blood, intestine and adipose tissues) will significantly reduce the cost-to-data output of our facilities which will benefit several groups involved in this renewal application.
The Core is located at the UCLA MacDonald Research Laboratories (MRL Building) and is fully equipped for molecular and analytical studies, including western blotting, immunohistochemistry and immunoassays. Equipments available include centrifuges, electrophoresis apparatus, ABI 7500 Fast sequence detector system for real-time RT-PCR, a FujiFilms LAS-4000 chemiluminescense reader for accurate densitometric assessment of protein expression on western immunoblots, , luminometer, spectrophotometer, fluorescent dye and 2 SpectraMax 96 well plate reader for ELISA analysis. The Core also has an EchoMRI for determination of small animal body constitution and is acquiring the Bio-plex 3D Suspension array System for fast and efficient marker expression and release analysis as well as high throughput mRNA expression and immunoassay measurements. In addition, several 4oC, -20oC, and -80oC refrigerators and freezers are available for sample storage with one -80o freezer designated specifically for human biopsy and human sample storage in order to cover the large number of clinical samples collected by members of this Program, in particular Project 1. In addition, the lab contains a liquid nitrogen tank, dark room, a 4oC/-20oC cold room, and a 37oC warm room. A cell and tissue culture room with 4 incubators and 4 biosafety cabinets, a fluorescence inverted microscope with fully equipped incubator for time-lapse imaging, a fluorescence upright microscope with DIC and apotome system for high resolution imaging, a NanoDrop Spectrophotometer for RNA and DNA concentration assessment are also available within Dr. Pothoulaki’s lab. We are also in the process of acquiring a Bioanalyzer for accurate determination of RNA quality.