A protocol for gene expression analysis of chondrocytes from bovine osteochondral plugs used for biotribological applications

摘要

class="kwd-title">Protocol name: Gene expression of bovine osteochondral plugs class="kwd-title">Keywords: RNA, Biotribology, Osteochondral plugs, Gene expression, Chondrocyte class="head no_bottom_margin" id="abs0010title">AbstractRNA isolation from human or animal cartilage tissue is necessary when performing mechanical or biotribological applications. Despite no influence on the cells and no alterations in gene expression patterns, enzymatic digestion of tissues should be avoided as it’s known that the expression of collagen 2 can be effected (Hayman et al., 2006 ). After mechanical or biotribological tests alternative options with an immediate disruption of the tissue should be contemplated. To obtain RNA, different tissue homogenization and disruption methods are available on the market (Yu et al., 2004 ), but not everyone is suitable for cartilage. Some of them neither homogenize the cartilage, while others are producing a lot of foam during disruption process. After trying some of the currently available methods, we chose the MagNA Lyser Instrument from Roche to disrupt the cartilage and further isolate RNA by using the Fibrous Tissue Kit from Qiagen. After RNA isolation, cDNA synthesis was performed by additionally adding RNA from bacteriophage MS2 for stabilization purposes. For the RTqPCR bovine primers were designed and tested for efficiency to confirm that the whole gene expression analysis is working. Our protocol explains a whole method to perform gene expression analysis from bovine cartilage, but can also be used for human or any other animal tissue.