Reference gene selection for transcriptional profiling by RT-qPCR in the 28-spotted larger potato ladybird

Abstract

• The stabilities of nine house-keeping genes are studied under four conditions. • RPL6 and RPL13 are for different developmental stages. • RPS18 and EF1α are for different tissues. • RPS18 and RPL13 are for four different experimental conditions. • Our findings establish an accurate normalization of qRT-PCR data. Henosepilachna vigintioctomaculata is one of the most serious defoliates attacking potatoes. However, studies on functional genes have greatly been limited due to the insufficiency of effective and stable endogenous references to normalize RT-qPCR data. In this report, nine housekeeping genes ( RPL4 , RPL6 , RPL13 , RPL32 , RPS18 , ACT , EF1α , GAPDH and α-TUB ) involved in different biological processes were selected. Their expression levels under diverse experimental conditions including developmental stages, tissues, temperatures and host plants were determined using RT-qPCR technology. The tested candidate genes were comprehensively ranked based on five alternative stability analysis methods (Ct value, geNorm, NormFinder, BestKeeper and ReFinder). The results revealed that the optimal internal reference genes varied under different experimental conditions. Any gene pair among the five candidates ( RPL4, RPL13, RPL32, RPS18 and EF1α ) was a suitable reference gene set under different temperatures and on different host plants. A combination of RPL6 and RPL13 was recommended as the best reference gene set across different developmental stages. A pair of RPS18 and EF1α was ranked as the optimal reference gene combination within different tissues. The most suitable reference genes were RPS18 and RPL13 under four different experimental conditions. Our findings not only establish an accurate and reliable normalization of RT-qPCR data, but also lay a solid foundation for further functional gene researches in H. vigintioctomaculata .