Screening of Reference Genes for Quantitative Real-Time PCR Analysis in Tissues and during Testis Development, and Application to Analyze the Expression of in (Teleostei, Cypriniformes, Cyprinidae).
The selection of proper reference genes is vital for ensuring precise quantitative real-time PCR (qPCR) assays. This study evaluates the stability of the expression of nine candidate reference genes in different tissues and during testicular development in . The results show that is recommended as a reference gene for qPCR analysis in tissues and during testicular development. Furthermore, we evaluated the optimal number of reference genes needed when calculating gene expression levels using the geomean method, revealing that two reference genes are sufficient. Specifically, and are recommended for analysis of gene expression in tissues, whereas and are advised for evaluating gene expression during testicular development. In addition, we examined the expression pattern of , a kinesin involved in the reshaping of spermatids. We detected peak expression levels of in testes, with its expression initially increasing before decreasing throughout testicular development. The highest expression of was observed in stage IV testes, the active period of spermiogenesis, suggesting a possible role for 1 in the regulation of the reshaping of spermatids and hence testicular development. This study represents the first investigation of reference genes for , providing a foundation for studying gene expression patterns and investigating gene expression regulation during testicular development.
