Validation of reference genes for expression studies during craniofacial development in arctic charr.
Validation of reference genes for expression studies during craniofacial development in arctic charr.
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DOI：
10.1371/journal.pone.0066389
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Jónsson ZO
中科院分区：
文献类型：
--
作者：
Ahi EP;Guðbrandsson J;Kapralova KH;Franzdóttir SR;Snorrason SS;Maier VH;Jónsson ZO
北极红点鲑 (Salvelinus alpinus) 是一种高度多态性的物种，在冰岛辛格瓦拉湖 (Lake Thingvallavatn) 中，已进化出四种表型形态。这些形态上的差异，特别是颅面结构的差异在胚胎发育过程中已经很明显，表明在颅面特征形成中重要的基因在不同形态之间的表达存在差异。为了生成检查北极红点鲑中这些表达差异的工具，本研究的目的是确定用于定量实时 PCR (qPCR) 的参考基因。具体目的是选择能够检测不同形态之间非常小的表达差异的参考基因。我们从文献中选择了 12 个候选参考基因，使用转录组测序 (RNA-seq) 获得的数据鉴定了相应的 charr 序列，并使用 qPCR 检查了它们的表达。发现许多候选参考基因稳定表达，但它们作为参考基因的质量排名根据所使用的分析类型而有很大差异。除了用于参考基因验证的常用软件之外，我们还使用经典统计学来评估表达谱，避免对具有相似表达模式（共同调节）的参考基因产生偏差。基于这些分析，我们选择了三个参考基因 ACTB、UB2L3 和 IF5A1 进行进一步评估。在三个已知颅面表达基因 sparc（或骨连接蛋白）、基质金属蛋白酶 2 (mmp2) 和 sox9（性别决定区 Y 框 9 蛋白）的表达研究中，使用 qPCR 在三个发育时间点的四个 charr 组衍生的胚胎头部中评估了它们的一致性。发现这三个参考基因非常适合研究形态型之间的表达差异，从而能够可靠地检测夏尔发育过程中较小的相对表达变化。此外，结果表明sparc和mmp2在不同北极红点鲑形态型的胚胎中存在差异表达。
Arctic charr (Salvelinus alpinus) is a highly polymorphic species and in Lake Thingvallavatn, Iceland, four phenotypic morphs have evolved. These differences in morphology, especially in craniofacial structures are already apparent during embryonic development, indicating that genes important in the formation of the craniofacial features are expressed differentially between the morphs. In order to generate tools to examine these expression differences in Arctic charr, the aim of the present study was to identify reference genes for quantitative real-time PCR (qPCR). The specific aim was to select reference genes which are able to detect very small expression differences among different morphs. We selected twelve candidate reference genes from the literature, identified corresponding charr sequences using data derived from transcriptome sequencing (RNA-seq) and examined their expression using qPCR. Many of the candidate reference genes were found to be stably expressed, yet their quality-rank as reference genes varied considerably depending on the type of analysis used. In addition to commonly used software for reference gene validation, we used classical statistics to evaluate expression profiles avoiding a bias for reference genes with similar expression patterns (co-regulation). Based on these analyses we chose three reference genes, ACTB, UB2L3 and IF5A1 for further evaluation. Their consistency was assessed in an expression study of three known craniofacially expressed genes, sparc (or osteonectin), matrix metalloprotease 2 (mmp2) and sox9 (sex-determining region Y box 9 protein) using qPCR in embryo heads derived from four charr groups at three developmental time points. The three reference genes were found to be very suitable for studying expression differences between the morphotypes, enabling robust detection of small relative expression changes during charr development. Further, the results showed that sparc and mmp2 are differentially expressed in embryos of different Arctic charr morphotypes.
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