Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis - Physiologie moléculaire et adaptation Accéder directement au contenu
Article Dans Une Revue Scientific Reports Année : 2018

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

Résumé

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. Several features make the African clawed frog, Xenopus laevis, an outstanding tool in biomedical research and vertebrate development. Since the 1930s, they have been used by doctors as a simple test for pregnancy. Egg production in female frogs is stimulated by the chorionic gonadotropin hormone found in pregnant woman's urine. Biologists have utilized this same method to induce female frogs to lay eggs and then fertilise in vitro for synchronised development 1. In turn, the synchronised development has been indispensable for developmental biologists to study regulatory and interactions networks that direct embryogenesis. The embryos develop externally and are free from direct maternal influences. This allows for embryos to be easily manipulated e.g. microinjections for genetic knockouts and knockdowns, germ layer dissections, tissue transplantations, etc. Rapid embryo growth means that a tadpole has fully functional set of organs within a couple of days. Coupled with a high number of brood size, X. laevis allows for large-scale genetic and chemical screens. Finally, the X. laevis genome has been fully sequenced and showed a remarkable structural similarity with the human genome 2,3. Real time quantitative polymerase chain reaction (RT-qPCR) is a widely popular technique for relative gene expression analysis, particularly during development. Reference genes within a RT-qPCR serve as internal controls for standard correction or normalisation 4. Ideal reference genes should be stable throughout the diverse experimental conditions. Their expression is expected to be under different regulatory mechanisms/pathways than the ones being investigated. However, no single reference genes have been shown to be universally constant during development. Furthermore, many studies have shown that traditionally used popular housekeeping genes used to normalize gene expression vary considerably under different conditions 5-8. There is currently only one study reporting the expression of reference genes for X. laevis 7 and another for its close relative, Xenopus tropicalis 8. In both cases, the reference genes characterised were the top most commonly used orthologues of genes found stably expressed in mammal tissues or were identified by systematic comparisons with traditional reference genes in mammals. These include the genes, glyceraldehyde-3-phosphate dehydrogenase (gadph),
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Dates et versions

hal-03919597 , version 1 (03-01-2023)

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Bilal B Mughal, Michelle Leemans, Petra Spirhanzlova, Barbara Demeneix, Jean-Baptiste Fini. Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis. Scientific Reports, 2018, 8 (1), pp.496. ⟨10.1038/s41598-017-18684-1⟩. ⟨hal-03919597⟩

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