Core Leader: Robert Raffai The lipoprotein, microvesjcle &lipid-free exRNA purification Core will serve all four sub-projects with the isolation and use of lipid-associated and lipid-free forms of exRNA carriers derived from human and mouse body fluids including plasma, as well as from conditioned cell culture medium and tissue extracts. The Core will interact closely with sub-project members to purify the desired types of exRNA-containing carriers including lipoproteins, cellular exosomes, tumor microvesicles, apoptotic bodies and lipid-free Ago2- complexes from provided fluid. Standardized methods will be established to efficiently isolate all forms of microvesicles from human and mouse plasma as well as from conditioned cell culture medium and to immunoprecipitate intact exRNAcomplexes of Ago2. This will include adapting published methods that,make use of serial centrifugation steps, size-exclusion and affinity chromatography, as well as FACS and immunoprecipitation. Quality control steps including western blot detection of microvesicle- and lipoprotein-associated proteins and RNA integrity measurements will be established to develop a standard operating procedure ensuring a reliable service for the efficient and reproducible isolation of desired exRNA carriers from biological fluids provided by all four sub-projects. The Core will work to provide reproducible preparations of desired subtypes of lipoproteins, cell-derived microvesicles and lipid-free Ago2-associated exRNA, from human and mouse plasma, as well as from conditioned cell culture medium and tissue extracts provided by the sub-projects. Additionally, the Core will actively collaborate with all sub-projects to assist in developing investigations into the biogenesis and cellular origins of lipid-associated and lipid-free forms ofexRNA in defined model systems. The overarching goal for the Core will be to provide an uninterrupted ahd streamlined service of exRNA isolation from fluids and tissue extracts to all four sub-projects to enable their experimental use as vehicles of intercellular communication in cell culture systems as well as in experimental models of human disease.
Supporting a single laboratory that is already fully equipped and experienced to purify lipid-associated exRNA carriers from biological fluids will avoid unnecessary duplication of resources and need for expertise that would othenwise be incurred should the isolations be performed by the individual sub-projects. Also, the uniformity of the isolated exRNA earners by the Core will provide consistency and reproducibility across assay systems, allowing for enhanced cross-fertilization among the sub-projects.
|Thomas, Molly F; L'Etoile, Noelle D; Ansel, K Mark (2014) Eri1: a conserved enzyme at the crossroads of multiple RNA-processing pathways. Trends Genet 30:298-307|