Selection and validation of reference genes for quantitative real-time PCR analysis across tissues at different...

Quantitative real-time polymerase chain reaction (qRT-PCR) is a highly sensitive and widely used method for analyzing gene expression profiles. Accurate qRT-PCR normalization requires the identification of stable reference genes under specific experimental conditions. Although seven reference genes have been used in Taraxacum kok-saghyz (TKS), an alternative natural rubber-producing crop, a systematic identification of reliable internal references for gene expression analysis across tissues at distinct developmental stages of TKS has not been conducted. In this study, we screened 12 candidate reference genes (CRGs) based on RNA-seq data from 26 TKS samples, representing five tissue types and nine developmental stages. The expression levels of the 12 CRGs, along with 7 previously reported reference genes (RRGs), were quantified by qRT-PCR across various tissues and developmental stages. The expression stability of the 19 genes was further evaluated by four commonly used algorithms (geNorm, NormFinder, comparative delta Ct, and BestKeeper), and their results were integrated by RefFinder to generate a comprehensive stability ranking. The final results revealed that TkADF1 and TkRPT6A were the most suitable internal control genes for the all-tissue group and leaf samples. TkUPL and TkSIZ1 were found to be optimal for root samples, while TkADF1 and TkSRPRA were preferred choices for latex samples. Moreover, validation using two rubber biosynthesis-related genes (TkFPS1 and TkSRPP2) confirmed the reliability of these recommended genes, showing a strong positive correlation with the RNA-seq data. This study provides reliable reference genes for qRT-PCR normalization in TKS, facilitating future research on developmental regulation and natural rubber biosynthesis.