Wuxi Fishery College, Nanjing Agricultural University, Wuxi 214081, China.
Fish Shellfish Immunol. 2012 Oct;33(4):775-9. doi: 10.1016/j.fsi.2012.06.027. Epub 2012 Jul 9.
Jian carp (Cyprinus carpio var. jian) is an important economic fish species cultured in China. In this report, we performed a systematic analysis to identify an appropriate housekeeping (HK) gene for the study of gene expression in Jian carp. For this purpose, partial DNA sequences of four potential candidate genes (elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate (GAPDH), beta-actin (ACTB), and 18S ribosomal RNA (18S rRNA) were isolated, and their expression levels were studied using RNA extracted from nine tissues (forebrain, hypothalamus, liver, fore-intestine, hind-intestine, ovary, muscle, heart, kidney) in juvenile and adult Jian carp. Gene expression levels were quantified by quantitative real time RT-PCR (qRT-PCR), and expression stability was evaluated by comparing the coefficients of variation (CV) of the Ct values. The results showed that EF-1α was the most suitable HK gene in all tissues of juvenile and adult Jian carp. However, at distinct juvenile and adult developmental stages, there was not a single optimal gene for normalization of expression levels in all tissues. EF-1α was the most stable gene only in forebrain, hypothalamus, liver, heart, and kidney. These results provide data that can be expected to aid gene expression analysis in Jian carp research, but underline the importance of identifying the optimal HK gene for each new experimental paradigm.
建鲤（Cyprinus carpio var. jian）是中国重要的养殖经济鱼类。在本报告中，我们进行了系统分析，以确定适合研究建鲤基因表达的内参（housekeeping，HK）基因。为此，我们分离了四个潜在候选基因（延伸因子 1α（elongation factor 1 alpha，EF-1α）、甘油醛-3-磷酸（glyceraldehyde-3-phosphate，GAPDH）、β-肌动蛋白（beta-actin，ACTB）和 18S 核糖体 RNA（18S ribosomal RNA，18S rRNA））的部分 DNA 序列，并使用从小麦中提取的 RNA 研究了它们在幼鱼和成年建鲤 9 种组织（前脑、下丘脑、肝脏、前肠、后肠、卵巢、肌肉、心脏、肾脏）中的表达水平。通过实时定量 RT-PCR（quantitative real time RT-PCR，qRT-PCR）定量基因表达水平，并通过比较 Ct 值的变异系数（coefficient of variation，CV）评估表达稳定性。结果表明，EF-1α是幼鱼和成年建鲤所有组织中最适合的 HK 基因。然而，在不同的幼鱼和成年发育阶段，没有一个单一的最佳基因可以在所有组织中归一化表达水平。EF-1α仅在前脑、下丘脑、肝脏、心脏和肾脏中是最稳定的基因。这些结果为基因表达分析提供了有价值的数据，但也强调了为每个新的实验范式确定最佳 HK 基因的重要性。
