Abstract
Real-time fluorescence quantitative PCR (RT-qPCR) is widely used to detect gene expression levels, and selection of reference genes is crucial to the accuracy of RT-qPCR results. Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that using the panel of reference genes for RT-qPCR is conducive to obtaining accurate experimental results. However, the selection of the panel of reference genes for RT-qPCR in rat developing cartilage has not been well documented. In this study, we selected eight reference genes commonly used in rat cartilage from literature (GAPDH, ACTB, 18S, GUSB, HPRT1, RPL4, RPL5, and SDHA) as candidates. Then, we screened out the optimal panel of reference genes in female and male rat cartilage of fetus (GD20), juvenile (PW6), and puberty (PW12) in physiology with stability analysis software of genes expression. Finally, we verified the reliability of the selected panel of reference genes with the rat model of intrauterine growth retardation (IUGR) induced by prenatal dexamethasone exposure (PDE). The results showed that the optimal panel of reference genes in cartilage at GD20, PW6, and PW12 in physiology was RPL4 + RPL5, which was consistent with the IUGR model, and there was no significant gender difference. Further, the results of standardizing the target genes showed that RPL4 + RPL5 performed smaller intragroup differences than other panels of reference genes or single reference genes. In conclusion, we found that the optimal panel of reference genes in female and male rat developing cartilage was RPL4 + RPL5, and there was no noticeable difference before and after birth.
Highlights
Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) has the advantages of high sensitivity, strong specificity, and high throughput, so it is widely used for the detection of gene expression levels (Nolan et al, 2006; Zhang et al, 2019)
This study explored the optimal panel of reference genes in the cartilage development period for the first time in physiology and prenatal dexamethasone exposure (PDE)-induced intrauterine growth retardation (IUGR) model, providing a methodological basis for studies related to cartilage development
The results showed that the circulation threshold (Ct) values of candidates in female rat articular cartilage were between 8 and 23 at GD20, PW6 and PW12 (Figure 1A)
Summary
Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) has the advantages of high sensitivity, strong specificity, and high throughput, so it is widely used for the detection of gene expression levels (Nolan et al, 2006; Zhang et al, 2019). Reference genes are the relatively constant genes for expression in tissues or cells, which can calibrate the experimental errors and ensure the accuracy of the experimental results. Researchers use reference genes for RTqPCR analysis to quantify the relative expression of the target genes. The selection of reference genes directly affects the accuracy of the experimental results (Bustin et al, 2009). Studies found that there was no generality in reference genes (de Oliveira et al, 2012; Pinto et al, 2012), and their stability varied by tissues, treatments, and developmental stages. Using a fixed reference gene often fails to accurately reflect the gene expression levels of various tissues, periods, and treatments (Sharan et al, 2015; Arya et al, 2017). It is necessary to select appropriate reference genes according to the model and actual situation
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