Identification and evaluation of reference genes for expression studies by RT-qPCR during embryonic development of the emerging model organism, Macrobrachium olfersii.

ABSTRACT

RT-qPCR is a sensitive and highly efficient technique that is widely used in gene expression analysis and to provide insight into the molecular mechanisms underlying embryonic development. The freshwater prawn, Macrobrachium olfersii is an emerging model organism, but, the stable reference genes of this species need to be identified and validated for RT-qPCR analysis. Thus, the aim of this study was to evaluate the expression stability of six genes (ß-act, GAPDH, EF-1α, RpL8, RpS6, AK) in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas) of M. olfersii. The expression stabilities of these genes were evaluated using geNorm, NormFinder, BestKeeper, ΔCt method and integrated tool RefFinder. In the general ranking, RpL8 and RpS6 were the most stable genes in embryos, while RpS6 and RpL8 were the most stable in a combined adult tissue analysis. Analysis of the adult tissues revealed that ß-act and AK were the most stable genes in cerebral ganglia, RpL8 and AK in muscle, and RpS6 and ß-act in hepatopancreas. EF-1α and GAPDH were the least stable genes and as normalizer genes in RT-qPCR affected expression of the Distal-less gene during M. olfersii development. This study provides suitable reference genes for RT-qPCR analysis and allows future studies of the gene expression in M. olfersii for understanding the molecular mechanisms of their development. To our knowledge, this is the first published study that identifies and evaluates reference genes for RT-qPCR analysis in M. olfersii and could be useful as basis for evaluations of reference genes in other prawns.