Validation of reference genes for gene expression analysis in melanin-injected black tiger shrimp (Penaeus monodon)

The expression stability of four potential reference genes (EF1-alpha, beta-actin, GAPDH and SubF0) was evaluated to normalize the expression of six immune-related genes (proPO1, proPO2, MnSOD, PAP, Rab7 and NOS) in different tissues of black tiger shrimp (Penaeus monodon) following melanin injection and using the geNorm and NormFinder algorithms. The stability rankings of the reference genes were as follows: EF1-alpha > beta-actin > SubF0 > GAPDH in the muscle, GAPDH > EF1-alpha > SubF0 > beta-actin in the carapace and EF1-alpha > GAPDH > beta-actin > SubF0 in the haemolymph. To normalize the expression of six immune-related genes of P. monodon in different tissues under the effect of melanin injection, EF1-alpha and GAPDH alone were the most suitable reference genes for the muscle and carapace, respectively, whereas the combination of EF1-alpha + beta-actin was the most suitable for the haemolymph. The expression of these six immune-related genes in P. monodon was upregulated in the haemolymph, downregulated in the carapace and nearly unchanged in the muscle of the melanin-injected group compared to that in the control group. Our results provide reference genes for reverse transcription quantitative polymerase chain reaction to analyse the protective effects of melanin in black tiger shrimp.