Evaluation of Suitable Reference Genes for Normalization of RT-qPCR in Echiura (Urechis unicinctus) during Developmental Process

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.