Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L.
Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L.
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DOI：
10.1016/j.aquaculture.2012.10.020
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发表时间：
2012-12-11
期刊：
影响因子：
4.5
通讯作者：
Juan Pazos, Antonio
中科院分区：
文献类型：
--
作者：
Mauriz, Oscar;Maneiro, Veronica;Juan Pazos, Antonio
对于基因表达的相对定量，靶基因的mRNA水平必须标准化。最常见的标准化策略是利用内参基因。然而，必须针对研究条件验证参考基因的稳定性。本工作的目的是选择最好的参考基因在双壳类软体动物Pecten maximus在生殖周期从一组七个候选人：肌动蛋白（act），18 S核糖体RNA（18 S），细胞色素c氧化酶亚基1（cox 1），真核翻译延伸因子1 α（ef 1 a），甘油醛-3-磷酸脱氢酶（gapdh），NADH脱氢酶（泛醌）1 α亚复合物亚基7（ndufa 7）和40 S核糖体蛋白SA（rpsa）。为了在生殖周期的不同阶段采集样本，在7个月的时间内，大约每月一次对3份样本进行采样。三种不同的统计模型（geNorm，Normalization和BestKeeper）已被用于分析通过逆转录定量真实的时间PCR（RT-qPCR）获得的结果。最合适的参考基因对每个组织都是特异性的。在大菱鲆卵巢中ndufa 7、rpsa和ef 1a是最稳定的基因。在大菱鲆精巢中，18 S、ndufa 7和gapdh是排序最高的参考基因。(C)2012年爱思唯尔B。V.保留所有权利。
For relative quantification of gene expression the mRNA levels of target genes must be normalized. The most common normalization strategy is the utilization of internal reference genes. However, the stability of reference genes must be validated for the conditions of the study. The aim of the present work is to select the best reference genes in the bivalve mollusc Pecten maximus during the reproductive cycle from a set of seven candidates: actin (act), 18S ribosomal RNA (18S), cytochrome c oxidase subunit 1 (cox1), eukaryotic translation elongation factor 1 alpha (ef1a), glyceraldehyde-3-phosphate dehydrogenase (gapdh), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 7 (ndufa7), and 40S ribosomal protein SA (rpsa). In order to have samples at different stages of the reproductive cycle three specimens were sampled at approximately monthly intervals during a period of 7 months. Three different statistical models (geNorm, NormFinder and BestKeeper) have been used to analyze the results obtained by reverse transcription quantitative real time PCR (RT-qPCR). The most suitable reference genes were specific to each tissue. In P. maximus ovary ndufa7, rpsa and ef1a were the most stable genes. In P. maximus testis 18S, ndufa7 and gapdh were the best ranked reference genes. (C) 2012 Elsevier B. V. All rights reserved.