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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 ,  β-actin , and  EF1-α ) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder,  EF1-α  and  GAPDH  ranked first and second as the most stable genes in testis groups whereas  GAPDH  and  EF1-α  were for ovaries from wild-caught broodstock and domesticated groups.  EF1-α  and  β-actin  ranked first and second for the eyestalk ablated ovaries. For geNorm,  EF1-α  and  GAPDH  had the best stability in all testis and ovaries from domesticated groups whereas  EF1-α  and  β-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-α,  the expected expression patterns were obtained in all cases. Therefore, this work suggests that  EF1-α  is more versatile as reference genes in qPCR analysis for reproductive system in  P. monodon .

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