Selection of reference genes suitable for normalization of qRT‐PCR data in spotted sea bass ( Lateolabrax maculatus ) under conditions of pathogen infections

Abstract

The aim of this study was to select the most suitable reference genes for qRT-PCR of spotted sea bass (Lateolabrax maculatus) under different pathogen infection conditions. A total of 10 candidate reference genes were selected; they included RPL19, RPL7, RPL13, TUGB, 18S rRNA, CYTB5, eIF5A, ACTB, GAB2 and GAPDH. Transcript levels of these candidate reference genes were examined using qRT-PCR analysis in 11 tissues (heart, liver, stomach, pectoral fin, intestine, muscle, brain, skin, gill, head kidney and spleen). Four algorithms, geNorm, NormFinder, BestKeeper and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. RPL19 was the most stable gene in different tissues in L. maculatus under normal conditions according to the overall ranking of the four algorithms. GAPDH was the most unstable gene in different tissues. GAB2 was the most stable gene in both head kidney and liver in L. maculatus under V. harveyi infection; RPL19 and RPL13 were the most stable genes in head kidney and liver in L. maculatus under S. iniae infection respectively; RPL19 and GAB2 were the most stable genes in head kidney and liver in L. maculatus under RGV infection respectively; RPL7 and RPL19 were the most stable genes in head kidney and spleen in L. maculatus under ISKNV infection respectively. GAPDH was the most unstable gene in immune tissues in L. maculatus under pathogen infections. The present study provides the first validated reference genes for accurate data normalization in transcript profiling in L. maculatus infected by bacteria (V. harveyi and S. iniae) and virus (ISKNV and RGV), which will be indispensable for further gene expression in this economically valuable marine teleost.