Abstract
When studying the altered expression of genes associated with cartilage regeneration by quantitative real-time RT-PCR (RT-qPCR), reference genes with highly stable expression during different stages of chondrocyte developmental are necessary to normalize gene expression accurately. Until now, no reports evaluating expression changes of commonly used reference genes in rabbit articular cartilage have been published. In this study, defects were made in rabbit articular cartilage, with or without insulin-like growth factor 1 (IGF-1) treatment, to create different chondrocyte living environments. The stability and intensity of the expressions of the candidate reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S Ribosomal RNA (18S rRNA), cyclophilin (CYP), hypoxanthine phosphoribosyl transferase (HPRT1), and β-2-microglobulin (B2M) were evaluated. The data were analyzed by geNorm and NormFinder. B2M and 18S rRNA were identified to be suitable reference genes for rabbit cartilage tissues.
Highlights
Many studies have focused on the process of repairing articular cartilage injuries
We examined the putative suitability of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, CYP, HPRT1 and B2M as reference genes in injury repair of articular cartilage tissue
The RT-qPCR expression data were analyzed using geNorm [21] and NormFinder [22] to identify the most suitable reference genes, namely, those that are minimally affected by chondrocyte development status
Summary
The various genes involved in this process are differentially expressed at different stages of repair. To ensure accurate and specific results, all of these variables must be controlled for thorough normalization, whereby the expression of a target gene is compared to that of a standard reference gene [5,6]. More and more evidence suggests that expression of some commonly accepted reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta actin (ACTB), are affected by experimental and clinical conditions; they may not always be suitable controls for normalization [7,8,9,10]. We examined the putative suitability of GAPDH, 18S rRNA, CYP, HPRT1 and B2M as reference genes in injury repair of articular cartilage tissue. The RT-qPCR expression data were analyzed using geNorm [21] and NormFinder [22] to identify the most suitable reference genes, namely, those that are minimally affected by chondrocyte development status
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