Identification and Validation of qRT-PCR Reference Genes for Analyzing Responses to High-Temperature Stress.

Quantitative real-time PCR (qRT-PCR) is an essential tool for analyzing and selecting stable reference genes. In order to screen for suitable reference genes under high-temperature stress conditions in , this study measured the relative expression levels of 17 candidate reference genes using qRT-PCR. Among these, four are traditional reference genes, while the remaining thirteen are candidate reference genes with no previous reports on their use as reference genes. The expression stability of the candidate reference gene expression was analyzed and evaluated using five methods: ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder. The results indicated that the and genes displayed the highest stability in expression under high-temperature stress conditions. To verify the stability of the reference genes, we treated with high-temperature stress, used the selected and as references, and analyzed the expression of the heat-responsive gene using qRT-PCR. The results showed that when and were used individually or in combination as reference genes, the relative expression of significantly increased and remained consistent under high-temperature treatment. This indicates that both and are suitable reference genes for qRT-PCR analysis in exposed to high-temperature stress. The novel reference genes identified in this study will serve as a reliable reference standard for gene expression studies in under high-temperature stress, thereby enhancing the accuracy and comparability of experimental data.