Christian Kirschneck1, Peter Proff2, Jochen Fanghänel3, Michael Wolf4, J Camilo Roldán5, Piero Römer6. 1. Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: christian.kirschneck@ukr.de. 2. Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: peter.proff@ukr.de. 3. Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: jochen.fanghaenel@ukr.de. 4. Department of Orthodontics, Rheinische Friedrich Wilhelm University of Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany. Electronic address: michael.wolf@ukb.uni-bonn.de. 5. 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. 6. Department of Orthodontics, University Medical Centre of Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053, Germany. Electronic address: piero.roemer@ukr.de.
Abstract
OBJECTIVES: 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. MATERIALS AND 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.
OBJECTIVES: 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. MATERIALS AND 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: Christian Niederau; Rogerio B Craveiro; Irma Azraq; Julia Brockhaus; Asisa Bastian; Christian Kirschneck; Michael Wolf Journal: Sci Rep Date: 2020-07-02 Impact factor: 4.379
Authors: Christian Kirschneck; Michael Maurer; Michael Wolf; Claudia Reicheneder; Peter Proff Journal: Int J Oral Sci Date: 2017-09 Impact factor: 6.344
Authors: Agnes Schröder; Paul Käppler; Ute Nazet; Jonathan Jantsch; Peter Proff; Fabian Cieplik; James Deschner; Christian Kirschneck Journal: Mediators Inflamm Date: 2020-04-28 Impact factor: 4.711