Stable Internal Reference Genes for Normalizing Real-Time Quantitative PCR in Baphicacanthus cusia under Hormonal Stimuli and UV Irradiation, and in Different Plant Organs

Yuxiang Huang, Hexin Tan, Jian Yu, Yue Chen, Zhiying Guo, Guoquan Wang, Qinglei Zhang, Junfeng Chen, Lei Zhang, Yong Diao

Baphicacanthus cusia (Nees) Bremek, the plant source for many kinds of drugs in traditional Chinese medicine, is widely distributed in South China, especially in Fujian. Recent studies about B. cusia mainly focus on its chemical composition and pharmacological effects, but further analysis of the plant's gene functions and expression is required to better understand the synthesis of its effective compounds. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful method for gene expression analysis. It is necessary to select a suitable reference gene for expression normalization to ensure the accuracy of RT-qPCR results. Ten candidate reference genes were selected from the transcriptome datasets of B. cusia in this study, and the expression stability was assessed across 60 samples representing different tissues and organs under various conditions, including ultraviolet (UV) irradiation, hormonal stimuli (jasmonic acid methyl ester and abscisic acid), and in different plant organs. By employing different algorithms, such as geNorm, NormFinder, and BestKeeper, which are complementary approaches based on different statistical procedures, 18S rRNA was found to be the most stable gene under UV irradiation and hormonal stimuli, whereas ubiquitin-conjugating enzyme E2 was the best suitable gene for different plant organs. This novel study aimed to screen for suitable reference genes and corresponding primer pairs specifically designed for gene expression studies in B. cusia, in particular for RT-qPCR analyses.