ArticleOsteoarthritis and cartilage2016
Importance of reference gene selection for articular cartilage mechanobiology studies.
Article in Osteoarthritis and cartilage, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 48 citations in OpenAlex.
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- Disuse atrophy of articular cartilage can be restored by mechanical reloading in mice.Molecular biology reports · 2024Article
- Effects of different combinations of mechanical loading intensity, duration, and frequency on the articular cartilage in mice.Molecular biology reports · 2024Article
- Screening and validation of the optimal panel of reference genes in colonic epithelium and relative cancer cell lines.Scientific reports · 2023Article
- Decreased SIRT1 Activity Is Involved in the Acute Injury Response of Chondrocytes to Ex Vivo Injurious Mechanical Overload.International journal of molecular sciences · 2023Article
- WNT16 Regulation of the Articular Chondrocyte Phenotype in Mice.Life (Basel, Switzerland) · 2023Article
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- Interferon-gamma modulates articular chondrocyte and osteoblast metabolism through protein kinase R-independent and dependent mechanisms.Biochemistry and biophysics reports · 2022Article
- Resveratrol regulates insulin resistance to improve the glycolytic pathway by activating SIRT2 in PCOS granulosa cells.Frontiers in nutrition · 2022Article
- A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads.Cells, tissues, organs · 2022Article
- Development of male-larger sexual size dimorphism in a lizard:Frontiers in physiology · 2022Article
- Elucidating mechano-pathology of osteoarthritis: transcriptome-wide differences in mechanically stressed aged human cartilage explants.Arthritis research & therapy · 2021Article
- Stability of housekeeping genes in inflamed joints of spontaneous and collagen-induced arthritis in DBA/1 mice.Inflammation research : official journal of the European Histamine Research Society ... [et al.] · 2021Article
- Rational, Unbiased Selection of Reference Genes for Pluripotent Stem Cell-Derived Cardiomyocytes.Tissue engineering. Part C, Methods · 2021Article
- Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces.The Journal of physiology · 2021Article
- Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage.Frontiers in genetics · 2020Article
- Article
- Human osteochondritis dissecans fragment-derived chondrocyte characteristics ex vivo, after monolayer expansion-induced de-differentiation, and after re-differentiation in alginate bead culture.BMC musculoskeletal disorders · 2018Article
- Evaluation of reference genes for gene expression studies in mouse and N2a cell ischemic stroke models using quantitative real-time PCR.BMC neuroscience · 2018Article
- Local Tensile Stress in the Development of Posttraumatic Osteoarthritis.BioMed research international · 2018Article
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
objectiveIdentification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively.
designThe expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder.
resultsAppropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions.
conclusionThis study demonstrates that commonly used 'reference genes' may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation.
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