Differential Gene Expression of Chondrocytes From a Porcine Impact Injury Model Using Suitable Reference Genes

INTRODUCTION: Impact injuries are a known risk factor for the development of osteoarthritis (OA). Identifying gene expression changes following an injury may help our understanding of early stage OA and identify targets for future intervention to slow disease progression. The objective of this study was to examine the gene expression changes of various genes implicated in OA following an injury to study the degenerative process in the tissue. An impaction to the articular surface of porcine patellae was used to initiate the degenerative process and quantitative real-time PCR (qPCR) was used to measure gene expression levels. Our goal was to determine the best housekeeping genes to normalize gene expression values for porcine articular cartilage and then evaluate changes in expression levels for several OA related genes following an impact injury. METHODS: Tissue Collection/Testing: Porcine knees were obtained from a slaughterhouse, the patellae sterilely removed, and subjected to one of three treatments: axial impaction (2000N load applied perpendicular to the cartilage surface), shear impaction (500N axial load with 10mm horizontal displacement to induce larger shear forces), or non-impacted control. Impactions were performed using a hydraulic load frame with a stainless impactor tip (10mm long by 10mm diameter). Following impactions, patellae were placed into culture to allow gene expression changes to be followed over time. Full thickness cartilage slices were cut from the impaction site after 0, 3, 7 or 14 days and stored at -80°C. RNA Extraction and qPCR: RNA was extracted using Tri Reagent followed by on-column DNase digestion. The ABI high capacity cDNA RT kit was used per the manufacturer’s protocol. Samples were analyzed with an iCycler IQ with SYBR Green master mix. Housekeeping Genes: Nine candidate housekeeping genes were selected from previous studies of porcine tissues: Beta-actin (actb), Beta-2-microglobulin (b2m1), Glyceraldehyde-3-phosphate dehydrogenase (gapdh), Hydroxymethylbilane synthase (hmbs), Ribosomal protein L4 (rpl4), Succinate dehydrogenase complex, subunit A (sdha), TATA box binding protein (tbp), Tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz) [1], and peptidylprolyl isomerase A (ppia) [2,3,4]. Ct values and PCR efficiency were determined for each gene and BestKeeper software [5], which uses pair-wise analysis and Pearson correlation coefficients to rank the genes, was used to determine the relative stability of the genes over the three treatments and four culture time points. OA Related Genes: Ten genes Ferritin, Heavy Polypeptide-1 (fth-1), Ferritin, Light Polypeptide (ftl), Collagen, Type I, Alpha 1 (col1a1) and Type II (col2a1), Matrix Metalloprotease-1 (mmp-3), TIMP Metallopeptidase Inhibitor-1 (timp-1) and 2 (timp-2), Chitinase 3-like 1 (chi3l1), Mitogen-inducible gene 6 (mig-6), and Chemokine ligand 16 (cxcl-16) were examined using qPCR at the 3 and 14 day time points. Data were analyzed using ANOVA and Tukey pairwise tests in JMP 7, and REST [6] to calculate fold changes (FCs). Results were normalized based on BestKeeper results and FC differences compared with α<0.1 considered to be trending towards significance and α<0.05 a significant difference. RESULTS: Gene stability was ranked, with genes expressing the highest BestKeeper correlation coefficient being the most stable (Figure 1). The