Selection and validation of stable reference genes for RT-qPCR analysis in Nepeta cataria L.

Nepeta cataria L. (catmint) is an important medicinal and aromatic plant valued for its bioactive secondary metabolites. Accurate RT-qPCR-based gene expression analysis requires stable reference genes for normalization. However, validated internal controls for this species are lacking. Although RT-qPCR workflows are standardized in many plants, reliance on commonly used housekeeping genes without species-specific validation can compromise accuracy. In this study, candidate reference genes were selected using available N. cataria transcriptomic information to ensure species-specific primer design and reliable gene identification. Ten genes were evaluated across four tissues (leaf, stem, root, and inflorescence) in ten breeding lines. Primer specificity was confirmed by semi-quantitative PCR, and expression stability was assessed using RT-qPCR followed by geNorm, NormFinder, BestKeeper, ΔCt method, and the integrative RefFinder platform. All analyses consistently identified tubulin beta chain (TUB) and Actin2 (ACT2) as the most stable genes, whereas UBE2E2 isoform X1 and GAPDH were the least stable. This transcriptome-guided and multi-algorithm validation strategy used here provides a robust framework that can be adapted to other plant species with available sequence resources supporting accurate normalization in future studies on secondary metabolism, stress responses, and molecular improvement.