Identification and Validation of Reliable Reference Genes for Gene Expression Studies in Koelreuteria paniculata

Abstract

RT-qPCR is considered a rapid and reliable technique for analyzing gene expression. This technique is commonly used to analyze the expression of various genes at diverse transcriptional levels in different samples. However, few studies have characterized ornamental Koelreuteria species for reliable reference genes. In this study, eight reference genes were evaluated as controls in RT-qPCR with SYBR green to quantify gene expression in different Koelreuteria paniculata samples. All selected reference genes showed a broad range of Ct values in all samples, which was supportive of their variable expression. Our results showed significant variation in the stable expression of K. paniculata genes. Sample data, analyzed using geNorm, NormFinder, and BestKeeper, showed that phospholipase (PLA2) and β-actin (ACT) were the most suitable and statistically reliable reference genes, whereas ribosomal protein L13 (RPL13) and elongation factor 1-α (EF1α) were less stable and unsuitable for use as internal controls. To compare gene expression levels, two or more reference genes should be used for data normalization. Thus, the stability and expression of both PLA2 and ACT were believed to provide better normalization and quantification of the transcript levels for gene expression studies in K. paniculata.