title: Odontoblast RNA stability in different temperature-based protocols for tooth storage.
abstract: OBJECTIVE
  To evaluate the effect of four tooth storage temperature-based methods on quality of RNA obtained from cells retrieved from human dental pulps and human pre-dentine.
  
  
  METHODS
  RNA was isolated from dental pulp tissue and from cells retrieved by scraping the pre-dentine of freshly extracted human third molars (n = 15) using TRIzol(®) reagent. Teeth were randomly assigned to the following temperature conditions: immediate RNA isolation after tooth extraction, liquid nitrogen (24 h), -80 °C (24 h), 20 °C (24 h) and 4 °C (6 h). RNA integrity was checked by the density of 28S and 18S ribosomal RNA. RT-PCR was used to analyse the expression of odontoblast makers (DSPP, DMP1 and MEPE) and the housekeeping gene GAPDH.
  
  
  RESULTS
  All experimental conditions evaluated preserved RNA integrity. The three odontoblastic markers were amplified from the pulp tissue and from the cells associated with pre-dentine.
  
  
  CONCLUSIONS
  The four storage options allowed RNA isolation for RT-PCR analysis. These findings may facilitate the use of clinically derived human dental pulp and odontoblasts for endodontic research.
authors.affiliation: Department of Restorative Dentistry, School of Dentistry, Federal University of Pelotas, Pelotas, Brazil.
authors.forename: M C M
authors.initials: MC
authors.lastname: Conde
authors.forename: F
authors.initials: F
authors.lastname: Nedel
authors.forename: V F
authors.initials: VF
authors.lastname: Campos
authors.forename: A J
authors.initials: AJ
authors.lastname: Smith
authors.forename: J E
authors.initials: JE
authors.lastname: Nör
authors.forename: F F
authors.initials: FF
authors.lastname: Demarco
authors.forename: S B C
authors.initials: SB
authors.lastname: Tarquinio
mesh_terms: D000293:Adolescent; D000328:Adult; D015925:Cryopreservation / Q000379:methods*; D003782:Dental Pulp / Q000166:cytology; D003804:Dentin / Q000166:cytology; D004587:Electrophoresis, Agar Gel; D016326:Extracellular Matrix Proteins / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D009804:Odontoblasts / Q000166:cytology*; D010750:Phosphoproteins / Q000032:analysis; D012313:RNA / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000032:analysis; D014021:Tissue Preservation / Q000379:methods*; D055815:Young Adult; D000099311:Dentin Sialophosphoprotein
chemical_list: D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D010750:Phosphoproteins; D012313:RNA; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein; C513309:DMP1 protein, human; C486579:MEPE protein, human