Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

Gene expression of reproductive system of the black tiger shrimp (Peneaus monodon) has been widely studied to address poor maturation problem in captivity. However, a systematic evaluation of reference genes in quantitative real-time PCR (qPCR) for P. monodon reproductive organs is lacking. In this study, the stability of four potential reference genes (18s rRNA, GAPDH, beta-actin, and EF1-alpha) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, EF1-alpha and GAPDH ranked first and second as the most stable genes in testis groups whereas GAPDH and EF1-alpha were for ovaries from wild-caught broodstock and domesticated groups. EF1-alpha and beta-actin ranked first and second for the eyestalk ablated ovaries. For geNorm, EF1-alpha and GAPDH had the best stability in all testis and ovaries from domesticated groups whereas EF1-alpha and beta-actin were the best for ovaries from wild-caught and eyestalk ablated groups. Moreover, the expression levels of two well-known reproductive genes, Dmc1 and Vitellogenin, were used to validate these reference genes. When normalized to EF1-alpha, the expected expression patterns were obtained in all cases. Therefore, this work suggests that EF1-alpha is more versatile as reference genes in qPCR analysis for reproductive system in P. monodon.