Screening and validation of suitable internal reference genes for gene expression analysis in Rubia cordifolia L. under different abiotic stresses

Rubia cordifolia L . is routinely employed as a traditional Chinese medicinal herb in clinical practice. As research on R. cordifolia advances, the identification of stable internal reference genes has become essential for gene expression studies. A comprehensive literature search reveals that the screening of internal reference genes for R. cordifolia has been reported neither domestically nor internationally. This study employed quantitative real-time PCR (RT-qPCR) to assess the expression stability of several candidate internal reference genes in R. cordifolia . The identification of internal reference genes that are stably expressed across different tissues and under various stress conditions in R. cordifolia has laid an important foundation for gene expression studies in this plant. This study profiled the expression stability of 12 candidate reference genes in R. cordifolia across various tissues and under multiple abiotic stresses (NaCl, dehydration, SA, MeJA, ABA) using four algorithms (geNorm, NormFinder, BestKeeper, ΔCt). The analysis identified hnRNP, UBQ, HIS3.3, PP2A , and PTBP2 as the most stable genes, while ACT7 and EF-1α were the least stable. RefFinder, which integrates the four algorithms, confirmed hnRNP, HIS3.3, PTBP2, PP2A , and RPL5 as the top five stable genes. Validation using the target gene MVK further confirmed the reliability of these reference genes. This study systematically evaluated the expression stability of internal reference genes in R. cordifolia , as a fundamental step toward reliable functional genomics research in this species. The findings pave the way for accurately elucidating the roles of its genes.