Abstract
Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus.
Highlights
Evolutionary origin of organs and comparative immunology of vertebrates[7,8]
Wang et al firstly evaluated the reliability of candidate reference genes (RGs) and found EF1A was a useful RG in different tissues of amphioxus, but number of candidate RGs and algorithms were few in the study[9]
Comparative analyses showed a high consistency for the ranking of stability for thirteen candidate RGs under each experimental condition among different statistical methods
Summary
Evolutionary origin of organs and comparative immunology of vertebrates[7,8]. Amphioxus is susceptible to antigen [e.g. stimulation of lipopolysaccharide (LPS) or bacteria], the same as vertebrates[9]. Ten tissue-specific transcriptomes of B. belcheri have been sequenced and our cooperators constructed online database (unpublished, http://wcy.pkusulab.com) It has been performed successfully for artificial feeding of amphioxus[11]. Further research is needed to evaluate more candidate RGs under more experimental conditions with more statistical algorithms, which will improve qRT-PCR reliability and accuracy for amphioxus studies. We aimed at identifying optimal RGs and recommending available normalization factors (NFs) for qRT-PCR analysis under different experimental conditions in amphioxus. Considering limits of economic, time and experimental materials in a real situation of lab, we analyzed correlation between optimal normalization factors (NFs) from geNorm algorithm and less NFs by using Pearson correlation coefficient (r) for each experimental condition, and recommended the reliable NFs and the least number of RGs to satisfy experiments in reality. Two target genes, encoding eyes absent protein (EYA) and hematopoietically expressed homeobox (HHEX) which are important during embryonic development of chordates[33,34], were chosen to determinate validation of selected RGs by three normalization strategies (recommended, optimal and the worst NFs) in different developmental stages of B. belcheri
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