title: Selection of reliable reference genes for qPCR studies on chondroprotective action.
abstract: BACKGROUND
  Chondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1beta-stimulated C-28/I2 chondrocyte model, using the geNorm software tool.
  
  
  RESULTS
  CPA treatment of C-28/I2 chondrocytes significantly affected the expression level of many reference genes (p < 0.05). According to their expression stability, geNorm analysis revealed rankings of the 3 most stable genes (from most stable to least stable) as follows: GAPDH, B2M and SDHA in glucosamine treated samples and HPRT1, GAPDH and B2M in curcumin or diacerein treated samples. Interestingly, ACTB was one of the most variably expressed genes throughout all experiments.
  
  
  CONCLUSIONS
  Our study points out the problem of relying on commonly used reference genes without an accurate validation process. For normalization purposes in gene profiling studies on glucosamine action, the genes GAPDH, B2M and SDHA are recommended as single reference genes depending on the expression level of the target gene or more favourably in combination. For experiments with curcumin and diacerein the use of HPRT1, GAPDH and B2M should be considered.
authors.affiliation: Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. stefan.toegel@univie.ac.at
authors.forename: Stefan
authors.initials: S
authors.lastname: Toegel
authors.forename: Wenwen
authors.initials: W
authors.lastname: Huang
authors.forename: Claudia
authors.initials: C
authors.lastname: Piana
authors.forename: Frank M
authors.initials: FM
authors.lastname: Unger
authors.forename: Michael
authors.initials: M
authors.lastname: Wirth
authors.forename: Mary B
authors.initials: MB
authors.lastname: Goldring
authors.forename: Franz
authors.initials: F
authors.lastname: Gabor
authors.forename: Helmut
authors.initials: H
authors.lastname: Viernstein
mesh_terms: D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D019902:Chondrocytes; D003474:Curcumin / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D005786:Gene Expression Regulation / Q000187:drug effects*; D005944:Glucosamine / Q000494:pharmacology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020011:Protective Agents / Q000494:pharmacology*; D012015:Reference Standards
chemical_list: D000880:Anthraquinones; D020011:Protective Agents; C025292:diacerein; D003474:Curcumin; D005944:Glucosamine