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 internal reference genes in R. cordifolia across various tissues and under multiple abiotic stresses (NaCl, dehydration, SA, MeJA, ABA), leveraging a suite of algorithms (GeNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder) for comprehensive evaluation.Analysis of the 12 candidate internal reference genes was performed using RefFinder, a computational tool that calculates a comprehensive stability ranking based on weighted geometric means. The collective analysis of candidate internal reference genes using geNorm, Normfinder, BestKeeper, and ΔCt methods identified hnRNP, UBQ, HIS3.3, PP2A , and PTBP2 as the five most stable genes. In contrast, ACT7 and EF-1α were found to be the least stable. Consistent with the findings from the four individual algorithms, RefFinder also identified hnRNP, HIS3.3, PTBP2, PP2A , and RPL5 as the top five most stable genes. Moreover,to further validate the reliability of the selected internal reference genes, we examined the expression patterns of the mevalonate kinase ( MVK) gene across a range of tissues and under a variety of treatment conditions, Using an inappropriate internal reference genes as a control can lead to significant bias in the results. 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.