BACKGROUND Flatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR) technique. Therefore, adequate reference genes for accurate normalization are required. RESULTS The stability of 12 potential reference genes was examined during larval development in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus) to determine the most suitable genes for qRT-PCR analysis. Transcription levels of genes encoding beta-Actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), annexin A2 (ANXA2), glutathione S-transferase (GST), ornithine decarboxylase (ODC), hypoxanthine phosphoribosyltransferase (HPRT1), ubiquitin (UBQ), elongation factor 1 alpha (eEF1A1), 18S ribosomal RNA, and the ribosomal proteins S4 (RPS4) and L13a (RPL13a) were quantitated. Two paralogous genes for ACTB were analyzed in each of both flatfish species. In addition, two paralogous genes for GAPDH were studied in Senegalese sole. RPL13a represented non-orthologous genes between both flatfish species. GeNorm and NormFinder analyses for expression stability revealed RPS4, UBQ and eEF1A1 as the most stable genes in Senegalese sole, Atlantic halibut and in a combined analysis. In all cases, paralogous genes exhibited differences in expression stability. CONCLUSION This work suggests RPS4, UBQ, and eEF1A1 genes as useful reference genes for accurate normalization in qRT-PCR studies in Senegalese sole and Atlantic halibut larvae. The congruent results between both species in spite of the drastic differences in larval development suggest that selected housekeeping genes (HKGs) could be useful in other flatfish species. However, the finding of paralogous gene copies differentially expressed during development in some HKGs underscores the necessity to identify orthologous genes.

中文翻译：

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使用实时 PCR 选择用于比目鱼幼虫基因表达研究的看家基因。

背景比目鱼变态涉及主要的生理和形态变化。由于其在水产养殖中的重要性以及作为发育研究的模型，一些基因表达研究已将重点放在使用定量实时 PCR (qRT-PCR) 技术对这一过程的理解上。因此，需要足够的参考基因进行准确的标准化。结果 在塞内加尔鲽 (Solea senegalensis) 和大西洋比目鱼 (Hippoglossus hippoglossus) 幼虫发育过程中检查了 12 个潜在参考基因的稳定性，以确定最适合 qRT-PCR 分析的基因。编码 β-肌动蛋白 (ACTB)、甘油醛-3P-脱氢酶 (GAPDH)、膜联蛋白 A2 (ANXA2)、谷胱甘肽 S-转移酶 (GST)、鸟氨酸脱羧酶 (ODC)、次黄嘌呤磷酸核糖基转移酶 (HPRT1)、对泛素 (UBQ)、延伸因子 1 α (eEF1A1)、18S 核糖体 RNA 以及核糖体蛋白 S4 (RPS4) 和 L13a (RPL13a) 进行定量。在两种比目鱼物种中的每一种中分析了 ACTB 的两个旁系同源基因。此外，在塞内加尔鞋底研究了 GAPDH 的两个旁系同源基因。RPL13a 代表两种比目鱼物种之间的非直系同源基因。GeNorm 和 NormFinder 对表达稳定性的分析显示 RPS4、UBQ 和 eEF1A1 是塞内加尔鲽、大西洋大比目鱼和联合分析中最稳定的基因。在所有情况下，旁系同源基因在表达稳定性方面表现出差异。结论 这项工作表明 RPS4、UBQ 和 eEF1A1 基因可作为有用的参考基因，用于在塞内加尔鲽和大西洋比目鱼幼体的 qRT-PCR 研究中进行准确标准化。尽管幼体发育存在巨大差异，但两种物种之间的一致结果表明，选定的管家基因 (HKG) 可能对其他比目鱼物种有用。然而，在某些 HKG 发育过程中发现的旁系同源基因拷贝差异表达强调了鉴定直系同源基因的必要性。