Abstract

Reverse transcription quantitative PCR (RT-qPCR) is commonly used for gene expression analysis. Appropriate reference genes enable the accurate quantification of gene expression. In this study, expressional stability of seven candidate reference genes in embryos and larvae of the Echiura Urechis unicinctus were evaluated using four programs, geNorm, NormFinder, BestKeeper, and ∆Ct method. The expressional stability was ATPase > eIF3 > EF-1-α > TUB > IDH > VDAC > ABL based on the comprehensive analysis, and the combination of ATPase, eIF3, and EF-1-α was predicted to be the most optimum choice for accurate gene quantification during the developmental process. Furthermore, we verified the competence of the candidate reference genes/combinations through evaluating the expression levels of TGF-β receptor 1 (TGFβR1) and SMAD4, and inspiringly found using either ATPase or eIF3 alone generated exactly similar results with that of using reference gene combinations. Therefore, we suggested that under certain circumstances, ATPase or eIF3 could be independently used as an alternative to quantify target gene expression in U. unicinctus during developmental process. This study provides important information for gene expression and function researches in Echiura or Annelida species.

中文翻译：

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适当的参考基因的正常化RT-qPCR在发育过程中的Echiura（Urechis unicinctus）的评价

摘要

逆转录定量PCR（RT-qPCR）通常用于基因表达分析。适当的参考基因可以准确定量基因表达。在这项研究中，在胚胎和螠虫动物门的幼虫7候选参考基因表现的稳定性，单环刺螠使用四个程序，geNorm，NormFinder，BestKeeper和ΔCT法进行评估。综合分析，表达稳定性为ATPase > eIF3 > EF-1- α> TUB > IDH > VDAC > ABL，并结合ATPase，eIF3和EF-1-预计α是在发育过程中进行准确基因定量的最佳选择。此外，我们通过评估TGF-β受体1（TGFβR1）和SMAD4的表达水平来验证候选参考基因/组合的能力，并令人鼓舞地发现，单独使用ATPase或eIF3产生的结果与使用参考基因​​组合的结果完全相似。因此，我们建议在某些情况下，可以将ATPase或eIF3独立用作量化U. unicinctus中靶基因表达的替代方法。在发展过程中。这项研究为棘壳类或An科物种的基因表达和功能研究提供了重要的信息。