Microarray Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathumthani, Thailand.
PLoS One. 2012;7(12):e52677. doi: 10.1371/journal.pone.0052677. Epub 2012 Dec 28.
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.
黑虎虾（Peneaus monodon）生殖系统的基因表达已被广泛研究，以解决其在人工养殖环境下成熟度低的问题。然而，针对黑虎虾生殖器官实时定量 PCR（qPCR）中参考基因的系统评估还很缺乏。在本研究中，利用生物信息学工具 NormFinder 和 geNorm，在不同条件下对生殖组织中的四个潜在参考基因（18s rRNA、GAPDH、β-actin 和 EF1-α）的稳定性进行了评估。对于 NormFinder，EF1-α和 GAPDH 在睾丸组中分别排名第一和第二，是最稳定的基因；而在野生亲虾和驯化组的卵巢中，GAPDH 和 EF1-α 则是最稳定的基因。对于切除眼柄的卵巢，EF1-α和 β-actin 排名第一和第二。对于 geNorm，EF1-α和 GAPDH 在所有驯化组的睾丸和卵巢中具有最佳的稳定性，而在野生亲虾和切除眼柄的卵巢中，EF1-α和 β-actin 是最稳定的基因。此外，使用两个已知的生殖基因 Dmc1 和卵黄蛋白原的表达水平来验证这些参考基因。当以 EF1-α 为内参时，在所有情况下都获得了预期的表达模式。因此，这项工作表明，EF1-α 作为黑虎虾生殖系统 qPCR 分析中的参考基因更加通用。
