Paria Anutosh, Dong Jie, Babu P P Suresh, Makesh M, Chaudhari Aparna, Thirunavukkarasu A R, Purushothaman C S, Rajendran K V
Indian J Exp Biol. 2016 Sep;54(9):597-605.
Quantitative real-time PCR (qRT-PCR), used to determine the gene expression profile, is an important tool in functional genomic research, including fishes. To obtain more robust and meaningful result, the best possible normalization of the data is of utmost significance. In the present study, we have evaluated the potential of five commonly used housekeeping genes i.e., elongation factor 1-α (EF1A), β-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-2-Microglobulin (B2M) in normal physiological conditions, developmental stages and in response to bacterial infection in Asian seabass, Lates calcarifer (Bloch), an important food fish cultured in the Asia-Pacific region. The expression levels of these five genes were estimated in 11 tissues of normal seabass juveniles, 14 embryonic and larval developmental stages and six tissues of Vibrio alginolyticus-challenged animals. Further, the expression stability of these genes was calculated based on three algorithms i.e. geNorm, NormFinder and BestKeeper. The results showed that although there are tissue-specific variations for each gene, ACTB and EF1A are the most stable genes across the tissues of normal animals. However, in bacteria-challenged animals, EF1A and 18S were found to be the best reference genes for data normalization. The expression of all the genes tested showed an increasing trend in developmental stages and the increase was significant at blastula stage. Among the five genes tested, EF1A and ACTB were found to be the genes with least variation and highest stability across the developmental stages. This forms the first report on validation of housekeeping genes in L. calcarifer, in the context of ontogenic development and in response to infection.
定量实时聚合酶链反应（qRT-PCR）用于确定基因表达谱，是功能基因组学研究（包括鱼类研究）中的一项重要工具。为了获得更可靠且有意义的结果，对数据进行尽可能最佳的标准化至关重要。在本研究中，我们评估了五个常用持家基因，即延伸因子1-α（EF1A）、β-肌动蛋白（ACTB）、18S核糖体RNA（18S）、甘油醛-3-磷酸脱氢酶（GAPDH）和β-2-微球蛋白（B2M）在正常生理条件、发育阶段以及在亚洲海鲈（尖吻鲈，Lates calcarifer (Bloch)，一种在亚太地区养殖的重要食用鱼）受到细菌感染时的潜力。在正常海鲈幼鱼的11个组织、14个胚胎和幼体发育阶段以及溶藻弧菌攻击的动物的6个组织中估计了这五个基因的表达水平。此外，基于geNorm、NormFinder和BestKeeper三种算法计算了这些基因的表达稳定性。结果表明，尽管每个基因都存在组织特异性差异，但ACTB和EF1A是正常动物组织中最稳定的基因。然而，在受到细菌攻击的动物中，EF1A和18S被发现是数据标准化的最佳参考基因。所有测试基因的表达在发育阶段呈上升趋势，并且在囊胚期显著增加。在测试的五个基因中，EF1A和ACTB在发育阶段变化最小且稳定性最高。这是关于尖吻鲈在个体发育和对感染反应方面持家基因验证的首篇报道。
