基于转录组数据筛选和评估赤点石斑鱼RT-qPCR分析的新参考基因

Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data.

作者信息

Wang Huan, Zhang Xiang, Liu Qiaohong, Liu Xiaochun, Ding Shaoxiong

机构信息

State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, Fujian, China.

State-Province Joint Engineering Laboratory of Marine Bioproducts and Technology, College of Ocean and Earth Sciences, Xiamen University, Xiamen, Fujian, China.

出版信息

PLoS One. 2017 Feb 9;12(2):e0171646. doi: 10.1371/journal.pone.0171646. eCollection 2017.

Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species.

石斑鱼是世界渔业市场上具有重要经济价值的鱼类品种。由于许多使用逆转录定量聚合酶链反应（RT-qPCR）的研究都涉及石斑鱼的基因表达，因此需要合适的内参基因来获得可靠且准确的结果。在本研究中，从最具价值的物种之一赤点石斑鱼的11个候选基因中，在一系列不同实验条件下鉴定出了最合适的内参基因。使用geNorm、NormFinder、BestKeeper和refFinder软件包，传统上使用的三个内参基因，β-肌动蛋白（β-ACT）、甘油醛-3-磷酸脱氢酶（GAPDH）和β2-微球蛋白（B2M），被确定不适用于赤点石斑鱼基因表达研究，而新鉴定出的两个内参基因，保守寡聚高尔基体复合体亚基5（Cog5）和布雷菲德菌素A抑制的鸟嘌呤核苷酸交换蛋白1（ARFGEF1），可在所有测试条件下通用。这些数据为在赤点石斑鱼和其他石斑鱼物种的基因表达RT-qPCR研究中获得更精确的结果奠定了基础。