A new reliable reference gene UBA52 for quantitative real-time polymerase chain reaction studies in pyloric cecal tissues of the starfish Asterias rubens.

Abstract

The starfish Asterias rubens is one of the most abundant echinoderm species in the White, Barents, North, and Baltic Seas. This species is an important component of marine ecosystems and a model object for certain biological studies, in particular those requiring quantitative estimation of gene expression. As a rule, expression at the transcriptional level is estimated by real-time qPCR using the ΔΔCt method, which allows the comparison of the copy number of target gene transcripts in samples with unknown mRNA/cDNA concentration. Application of this method requires normalization of the results relative to genes with stable expression levels (reference genes). The identification of reference genes is still a challenging task since data of this kind are missing for certain taxa, whereas the use of "standard" endogenous control genes without additional tests might lead to erroneous conclusions. We performed a preliminary analysis of the expression of many housekeeping genes in the pyloric ceca of A. rubens by high-throughput sequencing under normal and heat shock conditions. For one of them, the ubiquitin gene UBA52, low variation of expression (not greater than 2-fold) was shown using real-time qPCR. Tissues of pyloric ceca of normal adults and underyearlings and of adults after heat shock were used. The data obtained suggest that the UBA52 gene may be used as reference for normalization of gene expression at the mRNA level in the starfish A. rubens and probably in closely related species.