The development of RNA extraction protocols to examine the effects of early exercise on gene expression in the articular cartilage and subchondral bone of Perendale sheep : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Palmerston North, New Zealand

Abstract

To examine gene expression in vivo, total RNA was extracted from articular cartilage and subchondral bone. As limited methodology existed for ovine, RNA extraction was performed by optimization of previously published protocols used for other species and in vitro studies (Chomczynski & Mackey, 1995; Chomczynski & Sacchi, 1987; Heinrichs et al., 1997). Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the extracted RNA to evaluate the gene expression of inflammatory cytokines, collagens, collagenase and housekeeping genes glyceraldehyde phosphate dehydrogenase and Beta-Actin. We observed changes in the expression of inflammatory cytokines, collagen and collagenase genes between exercised and unexercised sheep. The results of this research are consistent with clinical imaging and microscopy studies which suggest that moderate exercise during early life can stimulate an adaptive response in articular cartilage and subchondral bone (Brama, Tekoppele, Bank, Barneveld, & van Weeren, 2000; Firth, 2006; Firth & Rogers, 2005b; Lammi et al., 1993). These changes can have a chondro-protective effect (Jones, Bennell, & Cicuttini, 2003; Otterness et al., 1998) and may reduce susceptibility to athletic injury in later life. Future research using fluorescent probes and polymerase chain reaction may permit quantification of gene expression in real-time to determine the anabolic and catabolic response of articular cartilage with developmental age and exercise in vivo.