Screening and evaluation of the stability of expression of reference genes in Lymantria dispar (Lepidoptera: Erebidae) using qRT-PCR.

The quantitative real-time polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantitative analysis of gene expression. Normalization of gene expression to that of relatively stably expressed housekeeping genes is required to facilitate the study of gene expression and to obtain more accurate RT-PCR data. However, no studies of the stability of expression of housekeeping genes in Lymantria dispar have been reported. In the present study, BestKeeper, GeNorm and NormFinder statistical software was used to evaluate the expression of thirteen candidate reference genes in L. dispar under different conditions. The expression levels of candidate reference genes were determined for two biological factors (developmental stages and tissues) and four abiotic treatments (temperature, insecticide, CO2 and starvation). The results showed that the best candidate reference genes in L. dispar were TUB, AK, RPS15 for developmental stages, RPL32 and GAPDH for tissues, ACTB and EF1-α for CO2 stress, GAPDH and RPL32 for temperature stress, RPS3 and GAPDH for insecticide stress, and GAPDH and RPS3 for starvation stress. In summary, EF1-α and TUB are preferential housekeeping genes in L. dispar under various conditions. These results provide a basis for the further study of functional genes of L. dispar.