Abstract
BackgroundExpression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes.ResultsTen candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin.ConclusionsBestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.
Highlights
Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results
The pig has been used as a model of human OA disease, cartilage repair, xenotransplantation, and gene transfer research, but no one has yet conducted a study to determine the ideal reference gene(s) for gene expression studies in porcine articular cartilage
In this study we propose to determine the best housekeeping genes for use in porcine articular cartilage and to evaluate three software packages, geNorm, BestKeeper, and NormFinder for determining overall gene stability
Summary
Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. Swingler et al [3] used sdha as a reference gene in their study of human OA cartilage These genes appeared to be selected based on literature, not selected based on evaluation of a panel of genes to identify the most stable gene. Pombo-Suarez et al [4] evaluated nine of these same reference genes in addition to ubiquitin C in human cartilage with advanced OA and found the rarely used housekeeping genes TATA box binding protein (tbp), ribosomal protein L13a (rpl13a) and beta-2-microglobulin (b2m) to be the most stably expressed genes while they found the most commonly used genes (gapdh, actb and 18s) to be the least stable. The pig has been used as a model of human OA disease, cartilage repair, xenotransplantation, and gene transfer research, but no one has yet conducted a study to determine the ideal reference gene(s) for gene expression studies in porcine articular cartilage
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