title: Validation of reference genes for real-time PCR of reproductive system in the black tiger shrimp.
abstract: 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.
authors.affiliation: Microarray Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathumthani, Thailand.
authors.forename: Rungnapa
authors.initials: R
authors.lastname: Leelatanawit
authors.forename: Amornpan
authors.initials: A
authors.lastname: Klanchui
authors.forename: Umaporn
authors.initials: U
authors.lastname: Uawisetwathana
authors.forename: Nitsara
authors.initials: N
authors.lastname: Karoonuthaisiri
mesh_terms: D000818:Animals; D033364:Decapoda / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D020043:Genes, Essential; D008297:Male; D010053:Ovary / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012098:Reproduction / Q000235:genetics; D013737:Testis / Q000378:metabolism