Selection of reference genes for accurate RT-qPCR analysis of dehydration tolerance in Xenopus laevis

Abstract

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a common technique for quantifying gene expression. The reliability and reproducibility of RT-qPCR relies heavily on normalization of target values to an internal control to account for experimental variability. Xenopus laevis, is well-known as a model in embryology and developmental biology but, with a sequenced genome, is now attracting attention as a natural model for studying environmental stress tolerance in adult amphibians. Specifically, recent studies have focused on gene responses to dehydration, however, a systematic screening of reference genes has yet to be done. Here, we evaluate the stability of eight candidate reference genes (actb.L, atp5bf1.L, canx.L, gapdh.S, rpl27.L, sdha.L, tbp.L, tbp.S) in eight tissues (brain, dorsal skin, heart, kidney, liver, lung, skeletal muscle, and ventral skin) using three popular algorithms (geNorm, Normfinder, and BestKeeper) in dehydrated X. laevis. A tissue-specific composite rank of each gene was determined from the stability rankings of each algorithm, and the optimal number of reference genes was determined by the pairwise-variation of normalization factors using the top n and n + 1 genes. The following reference gene sets were determined for each tissue: canx.L and rpl27.L in brain, rpl27.L and sdha.L in dorsal skin, actb.L and rpl27.L in heart, actb.L, gapdh.S, tbp.L, atp5f1b.L tbp.S and rpl27.L in kidney, sdha.L and atp5f1b.L in liver, actb.L and rpl27.L in lung, gapdh.S and rpl27.L in skeletal muscle, and rpl27.L and canx.L in ventral skin. The present study recommends that these genes be used as internal controls in future studies on dehydration tolerance in X. laevis.