title: Reference genes for valid gene expression studies on rat dental, periodontal and alveolar bone tissue by means of RT-qPCR with a focus on orthodontic tooth movement and periodontitis.
abstract: OBJECTIVE
  To obtain valid results in relative gene/mRNA-expression analyses by RT-qPCR, a careful selection of stable reference genes is required for normalization. Currently there is little information on reference gene stability in dental, periodontal and alveolar bone tissues of the rat, especially regarding orthodontic tooth movement and periodontitis. We therefore aimed to identify the best selection and number of reference genes under these experimental as well as physiological conditions.
  
  
  METHODS
  In 7 male Fischer344-rats the upper left first and second molars were moved orthodontically for 2 weeks and in 7 more animals additionally subjected to an experimental periodontitis, whereas 7 animals were left untreated. Tissue samples of defined size containing both molars (without crowns) as well as the adjacent periodontal and alveolar bone tissue were retrieved and RNA extracted for RT-qPCR analyses. Nine candidate reference genes were evaluated and ranked according to their expression stability by 4 different algorithms (geNorm, NormFinder, BestKeeper, comparative ΔCq).
  
  
  RESULTS
  PPIB/YWHAZ were the most stabile reference genes for the combined dental, periodontal and alveolar bone tissue of the rat overall, in untreated animals and rats with additional periodontitis, whereas PPIB/B2M performed best in orthodontically treated rats with YWHAZ ranking third. Gene-stability ranking differed considerably between investigated groups. A combination of two reference genes was found to be sufficient for normalization in all cases.
  
  
  CONCLUSIONS
  The substantial differences in expression stability emphasize the need for valid reference genes, when aiming for meaningful results in relative gene expression analyses. Our results should enable researchers to optimize gene expression analysis in future studies by choosing the most suitable reference genes for normalization.
authors.affiliation: Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: christian.kirschneck@ukr.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Kirschneck
authors.affiliation: Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: peter.proff@ukr.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Proff
authors.affiliation: Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: jochen.fanghaenel@ukr.de.
authors.forename: Jochen
authors.initials: J
authors.lastname: Fanghänel
authors.affiliation: Department of Orthodontics, Rheinische Friedrich Wilhelm University of Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany. Electronic address: michael.wolf@ukb.uni-bonn.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Wolf
authors.affiliation: Catholic Children's Hospital Wilhelmstift, Liliencronstraße 130, D-22149 Hamburg, Germany; Department of Craniomaxillofacial Surgery, University of Regensburg, Germany. Electronic address: jc.roldan@kkh-wilhelmstift.de.
authors.forename: J Camilo
authors.initials: JC
authors.lastname: Roldán
authors.affiliation: Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: piero.roemer@ukr.de.
authors.forename: Piero
authors.initials: P
authors.lastname: Römer
mesh_terms: D000465:Algorithms; D000539:Alveolar Process / Q000378:metabolism*; D000818:Animals; D015870:Gene Expression / Q000235:genetics*; D008297:Male; D008963:Molar / Q000033:anatomy & histology / Q000378:metabolism; D009970:Orthodontics; D010518:Periodontitis / Q000235:genetics* / Q000473:pathology; D010519:Periodontium / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D014070:Tooth / Q000378:metabolism*; D014087:Tooth Movement Techniques
chemical_list: D012313:RNA
keywords: Dental research; Gene expression; Gene expression regulation; Orthodontics; Periodontitis; RT-qPCR; Rats, Inbred F344