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Quantification of mRNA Using Real-Time PCR and Western Blot Analysis of MAPK Events in Chondrocyte/Agarose Constructs

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3D Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 695))

Abstract

In vitro models of chondrocyte mechanobiology have been used to compare the intracellular signalling pathways altered in normal and osteoarthritis-affected cartilage. However, differences in the model system and type of loading configuration have led to complicated pathways. This chapter is a follow-on of previous studies from our group utilising 3D agarose as a physiological model to study mechanotransduction pathways. Experimental methods are described to assess targets at the protein and gene expression level by Western blot analysis and real-time PCR, respectively. This chapter provides a quantitative gene expression approach to explore the intracellular pathways activated by both mechanical loading and inflammatory mediators and examine upstream phosphorylation events. Ultimately, development of methods used to analyse mechano-sensitive pathways will provide important information for the identification of appropriate pharmacological and physiotherapeutic agents for the treatment of osteoarthritis.

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Acknowledgements

Dr Chowdhury would like to thank Drs. Kerry Elliot, Lindsay Ramage, and Ying Zhou for their excellent support at the Queens Medical Research Institute, Edinburgh University. The protocols were developed with funding by the Wellcome Trust (project grant: 073972).

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Authors and Affiliations

  1. School of Engineering and Materials Science, Queen Mary University of London, London, UK

    David A. Lee, Oto-Ola Akanji & Tina T. Chowdhury

  2. Queens Medical Research Institute, Edinburgh University, Edinburgh, UK

    June Brand & Donald Salter

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  1. David A. Lee
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  2. June Brand
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  3. Donald Salter
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  4. Oto-Ola Akanji
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  5. Tina T. Chowdhury
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Editors and Affiliations

  1. Kroto Research Institute, Department of Engineering Materials, University of Sheffield, Broad Lane, Sheffield, S3 7HW, United Kingdom

    John W. Haycock

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Lee, D.A., Brand, J., Salter, D., Akanji, OO., Chowdhury, T.T. (2011). Quantification of mRNA Using Real-Time PCR and Western Blot Analysis of MAPK Events in Chondrocyte/Agarose Constructs. In: Haycock, J. (eds) 3D Cell Culture. Methods in Molecular Biology, vol 695. Humana Press. https://doi.org/10.1007/978-1-60761-984-0_6

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  • DOI: https://doi.org/10.1007/978-1-60761-984-0_6

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-983-3

  • Online ISBN: 978-1-60761-984-0

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