Global warming has emerged as the predominant trend in global climate change, with forecasts suggesting a rise in the occurrence and severity of extreme weather phenomena. As a result, ectothermic animals have become ideal model organisms for investigating the potential impacts of climate change on global biodiversity. Real-time fluorescence quantitative PCR (RT-qPCR) is a widely used technique for gene expression analysis. However, stable reference genes suitable for accurate normalization of transcriptional data across lizard samples have not yet been established. In this study, three skink species—Scincella modesta, S. reevesii, and Ateuchosaurus chinensis—were selected as model organisms to evaluate the expression stability of eight candidate housekeeping genes (18S rRNA, EF1α, ACTB, GAPDH, HPRT1, YWHAZ, RPS15, and RPS18) under nine distinct temperature conditions (6°C, 10°C, 14°C, 18°C, 22°C, 25°C, 28°C, 32°C, and 36°C). The Delta Ct method, BestKeeper, NormFinder, GeNorm, and RefFinder were utilized to assess expression consistency and to identify the most suitable reference genes, as well as the ideal number needed for reliable normalization. The results indicated that under varying temperature conditions, the best-performing internal controls were RPS18 and RPS15 for S. modesta; RPS18 and EF1α for S. reevesii; and RPS15 and YWHAZ for A. chinensis. Furthermore, RPS18, RPS15, and EF1α were identified as a stable and commonly applicable set of internal controls for RT-qPCR normalization across all three skink species and are recommended as reliable reference genes for use in related lizards exposed to thermal stress.

Global warming has emerged as the predominant trend in global climate change, with forecasts suggesting a rise in the occurrence and severity of extreme weather phenomena. As a result, ectothermic animals have become ideal model organisms for investigating the potential impacts of climate change on global biodiversity. Real-time fluorescence quantitative PCR (RT-qPCR) is a widely used technique for gene expression analysis. However, stable reference genes suitable for accurate normalization of transcriptional data across lizard samples have not yet been established. In this study, three skink species—Scincella modesta, S. reevesii, and Ateuchosaurus chinensis—were selected as model organisms to evaluate the expression stability of eight candidate housekeeping genes (18S rRNA, EF1α, ACTB, GAPDH, HPRT1, YWHAZ, RPS15, and RPS18) under nine distinct temperature conditions (6°C, 10°C, 14°C, 18°C, 22°C, 25°C, 28°C, 32°C, and 36°C). The Delta Ct method, BestKeeper, NormFinder, GeNorm, and RefFinder were utilized to assess expression consistency and to identify the most suitable reference genes, as well as the ideal number needed for reliable normalization. The results indicated that under varying temperature conditions, the best-performing internal controls were RPS18 and RPS15 for S. modesta; RPS18 and EF1α for S. reevesii; and RPS15 and YWHAZ for A. chinensis. Furthermore, RPS18, RPS15, and EF1α were identified as a stable and commonly applicable set of internal controls for RT-qPCR normalization across all three skink species and are recommended as reliable reference genes for use in related lizards exposed to thermal stress.