Selection and evaluation of reference genes for qRT-PCR analysis of different developmental stages in Chilo suppressalis

Abstract

• The stabilities of reference genes in different developmental stages and sexes of C. suppressalis were systematically studied. • H3 was the most stable reference gene for normalizing expression in different developmental stages. • It was not required to select and validate reference genes in different sexes of C. suppressalis . Quantitative real-time RT-PCR is considered to valuable method for the analysis of gene expression. But for the data accuracy of gene expression, the normalization of data using reference genes is essential. In this study, we focus on the analysis of the relative expression of nine commonly used candidate reference genes in the different developmental stages and female and male pupae/adults of Chilo suppressalis , and expression stabilities were analyzed by the ΔCt method, geNorm, NormFinder, and RefFinder. The results showed that H3 was the most stable reference genes in the different developmental stages while EF1 was the optimal genes in pupae/adult of different sexes of C. suppressalis . To validate, the expression profile of the gene encoding heat shock protein 60 ( Hsp60 ) was investigated. The results showed that the transcription of target gene Hsp60 varied depending on the reference gene selected for normalization in different developmental stages, but the trend of expression of Hsp60 between the most stable reference gene combination and the least stable reference gene in pupae/adults of different sexes was the same. Therefore, it is imperative to select the reference genes to estimate target gene expression accurately in the specific experiment. This study provides scientific basis on functional analyses of the important genes contributed to the development and sex differentiation of C. suppressalis .