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Sci Rep January 1, 2018; 8 (1): 496.

Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis.

Mughal BB , Leemans M , Spirhanzlova P , Demeneix B , Fini JB .

Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are eef1a1, odc1, rpl8, and actnB, although there is a lack of ad hoc reference genes for X. laevis. Here, we used previously published RNA-seq data on different X. laevis stages and identified the top 14 candidate genes with respect to their expression levels as a function of developmental stage and degree of variation. We further evaluated the stability of these and other candidate genes using RT-qPCR on various stages including the unfertilised eggs, whole embryos during early development and brains during late development. We used four different statistical software packages: deltaCT, geNorm, NormFinder and BestKeeper. We report optimized reference gene pair combinations for studying development (early whole embryos), brains at later stages (metamorphosis and adult), and  thyroid signalling. These reference gene pairs are suitable for studying different aspects of X. laevis development and organogenesis.

PubMed ID: 29323148
PMC ID: PMC5764961
Article link: Sci Rep

Species referenced: Xenopus laevis
Genes referenced: actb cfl1 clta cox7b dio1 dio2 dio3 eef1a1 gapdh lpcat1 lpcat3 mcts1 mlf2 mtch2 odc1 ppp1ca prcp ralb rpl8 slc35b1 sub1 thra thrb tm9sf4 ube2m
GO keywords: thyroid hormone mediated signaling pathway [+]

Article Images: [+] show captions
References [+] :