title: Evaluation of methods for the isolation of high quality RNA from bovine and cervine hide biopsies.
abstract: Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843-4467, USA.
authors.forename: Jaime L
authors.initials: JL
authors.lastname: Brannan
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Holman
authors.forename: Pia U
authors.initials: PU
authors.lastname: Olafson
authors.forename: John H
authors.initials: JH
authors.lastname: Pruett
authors.forename: Penny K
authors.initials: PK
authors.lastname: Riggs
mesh_terms: D000818:Animals; D001707:Biopsy, Needle / Q000662:veterinary; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology / Q000469:parasitology* / Q000473:pathology; D015925:Cryopreservation / Q000662:veterinary; D003670:Deer / Q000469:parasitology*; D004202:Disinfectants; D004478:Ectoparasitic Infestations / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*; D019075:Electrophoresis, Capillary / Q000662:veterinary; D005260:Female; D006146:Guanidines; D012313:RNA / Q000302:isolation & purification* / Q000592:standards; D011933:Reagent Kits, Diagnostic / Q000662:veterinary; D012867:Skin / Q000737:chemistry / Q000469:parasitology / Q000473:pathology*; D013053:Spectrophotometry / Q000662:veterinary; D013861:Thiocyanates
chemical_list: D004202:Disinfectants; D006146:Guanidines; D011933:Reagent Kits, Diagnostic; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012313:RNA