Validation of housekeeping genes for gene expression studies in western corn rootworm (Diabrotica virgifera virgifera)

Real-time quantitative PCR (qRT-PCR) is a powerful technique for the investigation of comparative gene expression. To normalize results, use of a highly stable housekeeping gene (HKG) as an internal control is recommended. However, there are several reports suggesting that housekeeping genes can be regulated under different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The genes expression studies for WCR are being conducted for some time, however, a study to identify the stable reference genes under different conditions in this important pest is lacking. Using four distinct algorithms (Genorm, Normfinder, Bestkeeper and Delta CT), we evaluated five candidate HKG genes (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) to determine the most efficient reference genes in four different experiments (different tissues; different developmental stages; dsRNA exposures; and Bt toxin exposures). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. β-actin showed the high stable expression among different stages, RPS9 showed the high stable expression in dsRNA experiment, and both experiments showed EF1a as the second most stable gene. Also, EF1a was the most stable gene in Bt and different tissues experiments. Therefore, we recommend EF1a as a suitable HKG for efficient normalization among WCR experiments, although it should be better if we can identify and use a HKG experiment-by-experiment. These results will enable a more accurate and reliable normalization of qRT-PCR data in WCR.