Selection of reference genes for normalization of quantitative real-time polymerase chain reaction analysis in...

Aulacorthum solani (Hemiptera: Aphididae) has emerged as a significant global economic pest threatening diverse agricultural crops. Quantitative real-time polymerase chain reaction is a fundamental tool in molecular biology for quantifying gene expression, with its accuracy heavily reliant on the selection of stable reference genes under specific experimental conditions. However, no studies have identified suitable reference genes for A. solani across varying experimental contexts. In this study, 6 candidate reference genes including 40S ribosomal protein S12 (RPS12), 40S ribosomal protein S18 (RPS18), TATA-box-binding protein (TBP), alpha tubulin (α-Tub), beta tubulin (β-Tub), and 60S ribosomal protein L27 (RPL27), were evaluated for expression stability under 6 distinct experimental conditions to normalize quantitative real-time polymerase chain reaction data. Gene expression stability was assessed using the ΔCt method, GeNorm, BestKeeper, NormFinder, and RefFinder. Results indicated that β-Tub and RPL27 exhibited the highest stability across developmental stages of A. solani, while α-Tub and RPS12 were most reliable for studies involving wing dimorphism. For tissue-specific analyses, β-Tub and RPS18 showed optimal expression consistency. Under different temperatures, RPS18 and α-Tub were identified as the most suitable reference genes. In response to insecticide exposure, TBP and α-Tub were recommended, whereas under different photoperiod regimes, RPL27 and α-Tub demonstrated the most stable expression. This study provides a validated set of reference genes to improve the accuracy and reliability of gene expression analysis and functional genomic research in A. solani.