title: A rapid, reliable method to isolate high quality endothelial RNA from small spatially-defined locations.
abstract: Phenotypic heterogeneity of the endothelium has important implications for cell sourcing for cardiovascular tissue engineered devices and is fundamental to many cardiovascular diseases. A critical first step to identifying genetic regulators associated with particular endothelial phenotypes is reliable isolation of pure RNA from the cell subpopulations of interest. We present here a rapid method for the isolation of endothelial RNA from small spatially-defined locations, illustrated for two sides of the porcine aortic valve. Endothelial cells were retrieved from fresh tissue by freezing them to a glass substrate, from which they were lysed in guanidine thiocyanate buffer for RNA isolation. Valve endothelial cells isolated by this technique stained positively for CD31 and von Willebrand factor, consistent with an endothelial phenotype, with no evidence of contamination by alpha-smooth muscle actin-positive valve interstitial cells or CD45-positive leukocytes. RNA integrity was excellent in 80% of the samples, with over 100 ng of total RNA typically obtained from each side of the valve. This rapid method yields high quality pure endothelial RNA in sufficient quantities for amplification and subsequent use in device-, cell-, and location-specific transcriptional profiling by microarray technologies, and thus facilitates studies of spatial gene regulation.
authors.affiliation: Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104-6383, USA. simmons2@mail.med.upenn.edu
authors.forename: Craig A
authors.initials: CA
authors.lastname: Simmons
authors.forename: Jenny
authors.initials: J
authors.lastname: Zilberberg
authors.forename: Peter F
authors.initials: PF
authors.lastname: Davies
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003080:Cold Temperature; D042783:Endothelial Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D006146:Guanidines / Q000737:chemistry; D046228:Microarray Analysis / Q000379:methods*; D008832:Microchemistry / Q000379:methods*; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods*; D013552:Swine; D013861:Thiocyanates / Q000737:chemistry; D014018:Tissue Distribution; D014021:Tissue Preservation / Q000379:methods*
chemical_list: D006146:Guanidines; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012313:RNA