Quantification of mRNA using real-time PCR and Western blot analysis of MAPK events in chondrocyte/agarose constructs

David A Lee, June Brand, Donald Salter, Oto-Ola Akanji, Tina T Chowdhury

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)77-97
Number of pages21
JournalMethods in Molecular Biology
Volume695
DOIs
Publication statusPublished - 2011

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