Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer Chilo sacchariphagus (Lepidoptera: Pyralidae).

Simple Summary: Gene expression analysis by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) can provide strong evidence for scientists to understand the molecular mechanisms underlying various physiological processes. Selecting appropriate reference genes under specific experimental conditions are prerequisites for achieving the accurate results of qRT-PCR. Here, the expression stability of seven reference genes was evaluated in Chilo sacchariphagus, a destructive pest of sugarcane, under different experimental conditions, encompassing tissues, temperatures, and sexes. The expression patterns of C. sacchariphagus pheromone binding protein 1 gene (CsacPBP1) across three different experimental conditions mentioned above were evaluated to verify the results. The findings of this study will lay an important foundation for the future study on functional gene expressions in the pest. Chilo sacchariphagus (Lepidoptera: Pyralidae) is an economically important sugarcane pest. Although numerous studies were conducted on the physiological responses in C. sacchariphagus, little is known regarding the genes regulating these physiological processes. Gene expression analysis by qRT-PCR can offer a significant indication for functional gene studies. To our knowledge, the reference genes of C. sacchariphagus have not been screened or evaluated, which hinders the functional gene study. In the present study, the stability of seven reference genes (β-ACT, GAPDH, BTF3, 28S, RPL7, EF1α, and SDHA) was evaluated in C. sacchariphagus under different experimental conditions, including tissues (antenna, head, thorax, abdomen, leg, and wing), temperatures (4 °C, 25 °C, and 37 °C) and sexes (male and female), through RefFinder, which integrates four algorithms (Normfinder, BestKeeper, ΔCt method, and geNorm). The findings suggested that the combination of β-ACT and RPL7 is ideal to analyze gene expressions in different tissues and at distinct temperatures, and EF1α and SDHA were suitable reference genes for comparing gene expressions between sexes. Finally, the expression profiles of CsacPBP1 gene were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. This study represents the first kind in screening out suitable reference genes for gene expression analysis in C. sacchariphagus. Information from this study is poised to galvanize future inquiry into the gene expression of C. sacchariphagus, an economically important pest of sugarcane. [ABSTRACT FROM AUTHOR]