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

Abstract

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.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development

“…In previous studies, CRISPR/Cas9 gRNA was injected into zygote/1-cell stage porcine embryos to modify genes that are not essential for pre- or post-implantation development ( Whitworth et al, 2017 ). However, Uba52 is classified as a housekeeping gene ( Ahn et al, 2008 ; Sadritdinova et al, 2014 ; Schoen et al, 2015 ) and is likely activated before major embryonic genome activation ( Latham and Schultz, 2001 ; Østrup et al, 2013 ). Thus, the injection time window had to be moved to metaphase II stage oocyte, i.e.…”

“…While the polyubiquitin genes play a key role in stress-responses such as heat shock, starvation ( Finley et al, 1987 ; Fornace et al, 1989 ), DNA damage, oxidative stress ( Vihervaara et al, 2013 ) and heavy metal cytotoxicity response ( Lee et al, 2015 ), the ubiquitin-ribosomal fusion genes are stably expressed to satisfy the demand for ubiquitin under basal conditions ( Bianchi et al, 2015 ). The Uba52 gene has been identified as a housekeeping gene ( Warrington et al, 2000 ) and the transcript has been used as a reference in rhesus monkey ( Ahn et al, 2008 ), starfish ( Sadritdinova et al, 2014 ) and bovine tissues ( Schoen et al, 2015 ), to quantify gene expression. Through cDNA microarray analysis of gene expression in porcine oocytes and early preimplantation embryos, Uba52 expression in the blastocyst stage embryo was six times higher than the metaphase II oocytes ( Whitworth et al, 2005 ).…”