Evaluation of internal control genes for qRT-PCR normalization in tissues and cell culture for antiviral studies of grass carp (Ctenopharyngodon idella)

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most commonly used techniques for RNA expression. To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. Here, we cloned partial sequence of elongation factor 1α (EF1α) gene from grass carp (Ctenopharyngodon idella). The stabilities of four commonly used internal control genes encoding 18S rRNA, β-actin, EF1α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were integratedly assessed using the geNorm, NormFinder and BestKeeper programs. Integrative analyses of qRT-PCR data indicated that the stability ranking of the examined genes was 18S rRNA > EF1α > GAPDH > β-actin in gill, head kidney, heart, intestine, liver, muscle, skin, spleen, and trunk kidney tissues in untreated grass carp. When the same calculations were done in spleen tissue at different time points post grass carp reovirus (GCRV) infection, the gene ranking was 18S rRNA > β-actin > GAPDH > EF1α. The rank ordering of expression stability was EF1α > β-actin > 18S rRNA > GAPDH in C. idella kidney (CIK) cell culture stimulated by poly(I:C). The recommended ranking was EF1α > GAPDH > β-actin > 18S rRNA in CIK cells infected by GCRV. The results indicated that 18S rRNA was the best invariant internal control gene in individual level in grass carp, EF1α was the most suitable in CIK cell culture stimulated by poly(I:C) or infected by GCRV. As an assay, EF1α was employed to examine the changes of Toll-like receptor 3 (TLR3) and melanoma differentiation associated gene 5 (MDA5) after virus infection in CIK cells. These data laid the foundation for more precise results in qRT-PCR studies of gene expression in grass carp.

Research highlights► Optimal internal control gene serves the more accurate expressional and functional studies. ► The stabilities of the most commonly used internal control genes were evaluated in grass carp. ► Three software programs were employed to ascertain the most suitable internal control gene to normalize the RNA load in antiviral immunity studies in grass carp. ► Determine the stable orders of internal control genes according to the total scores of three software programs.