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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.

Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus