Identification and Validation of Reference Genes for Reliable RT-qPCR Normalization in Schisandra chinensis Across...

Reverse transcription quantitative real-time PCR (RT-qPCR) is a highly efficient and sensitive technique for quantifying gene transcript levels. The accuracy of gene expression analysis depends critically on the selection of appropriate reference genes for normalization, which is essential to minimize technical variation arising from differences in RNA quality, reverse transcription efficiency, and sample handling. Schisandra chinensis is a medicinally important plant with a long history of use in traditional Chinese medicine and has gained increasing global recognition. In recent years, a growing number of studies have employed molecular biology approaches to investigate the molecular mechanisms underlying secondary metabolite biosynthesis in S. chinensis. However, systematically validated reference genes for RT-qPCR analysis in this species have not yet been established. In the present study, the expression stability of eleven candidate reference genes was evaluated across different tissues and under various abiotic stress conditions in S. chinensis using four statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Comprehensive analysis revealed that PP2A15 and UBC2 were the optimal reference gene combination for leaves; UBC2 and UBC11 for stems; RPL6 and PP2A15 for roots; RPL21 and RPL6 for fruits; and RPL6 and UBC11 as the best-performing pair across all tissue types. Under abiotic stress conditions, UBC11 and UBC2 exhibited the highest stability in both leaves and roots under salt stress; UBC2 and GPN1 proved most stable under alkaline stress; UBC2 and RPL6 were identified as the most suitable combination under drought stress; and UBC2 and UBQ12 demonstrated consistently stable expression across all three abiotic stress treatments. The reliability of these reference gene combinations was further validated by examining the expression profiles of three target genes. Collectively, these findings establish a validated reference gene toolkit for future gene expression studies in S. chinensis, particularly for the functional characterization of genes involved in lignan biosynthesis and abiotic stress responses.