Identification of Appropriate Reference Genes for Gene Expression Studies by Quantitative Real-time PCR in Tribolium castaneum After Exposure to Phosphine
Abstract：The Real-time quantitative PCR (qRT-PCR) is a common approach to analyse the gene expression level, in order to obtain reliable experimental data, it is critical to select an appropriate reference gene. However, there has been little research on the insect reference genes. This research was conducted to determine stable referece gene(s) for normalization in future expression studies on the red flour beetle (Tribolium castaneum). The expressions of candidate reference genes β-actin, GAPDH, α-Tubulin, SYN1, SYN6, RPS3, RPS18 and RPL13a in Tribolium castaneum after exposure to the phosphine were detected by qRT-PCR using relative quantification. According to the qRT-PCR reaction result, these eight candidate reference genes were all specifically amplified by the primers pairs with high efficiency. Then the expression stability of these genes was analysed by geNorm and NormFinder software. Based on the results of geNorm analysis, the stability of the eight candidate reference genes from high to low was RPS18 =RPL13a (0.007)＞SYN6(0.015)＞RPS3(0.038)＞β-actin (0.044)＞α-Tubulin(0.105)＞SYN1(0.154)＞GAPDH (0.205), and V2/3 value of pairwise variances analysis of eight candidate reference genes was 0.006. The results showed that RPS18 and RPL13a were the most suitable reference genes in T. castaneum after exposure to the phosphine and the appropriate number of reference genes was 2, whereas GAPDH, α-Tubulin and β-actin which have a wide range of application were poorly ranked. On the other hand, based on the results of the NormFinder analysis, the stability of eight candidate reference genes from high to low was RPS18 (0.002)＞RPL13a(0.016)＞β-actin (0.042)＞RPS3 (0.044)＞SYN6(0.055)＞α-Tubulin (0.066)＞SYN1(0.077)＞GAPDH (0.126). The results showed that RPS18 was the most suitable reference gene, and the best combination of two genes were RPS18 and RPL13a. All above results suggest that RPS18 and RPL13a are suitable reference genes and can be used to normalize mRNA levels in different strains of T. castaneum after exposure to phosphine . This work is contributable to lay a foundation for future gene expression studies in the T. castaneum, and may also act as a resource to screen reference genes for expression studies in other insects.
