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

Bilal B Mughal,Petra Spirhanzlova,Jean-Baptiste Fini,Michelle Leemans,Barbara Demeneix

doi:10.1038/s41598-017-18684-1

Bilal B Mughal, Petra Spirhanzlova + Show 3 more

Open Access

https://doi.org/10.1038/s41598-017-18684-1

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Abstract

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.

Highlights

Several features make the African clawed frog, Xenopus laevis, an outstanding tool in biomedical research and vertebrate development
Several reports demonstrate that these choices can lead to inadequate normalization of gene expression. gadph, actb and h4 have been shown to be differentially expressed during the pre- and post-natal periods in mammals[9,10,11]
We have identified and characterised suitable reference genes for studying development, brains at later stages, and thyroid signalling in X. laevis

Summary

Introduction

Several features make the African clawed frog, Xenopus laevis, an outstanding tool in biomedical research and vertebrate development. Deep RNA sequencing (RNA-seq) has become a powerful tool in high-throughput transcriptomic studies with its high resolution, large data set, and sensitivity This technique has been used to identify novel reference genes for model systems such as the human cell cultures, zebrafish, mice and plants[12,13,14,15]. We used the recent RNA-seq data from Session et al, to identify the most stably expressed genes during X. laevis development[3] Their expression stability was further validated using RT-qPCR. We used four statistical algorithms, delta-CT16, geNorm[17], BestKeeper[18] and NormFinder[19] According to their stability, we identified the best reference genes candidates during different stages of X. laevis development. We further identified and validated the reference gene candidates for stage NF48 X. laevis brains when studying thyroid hormone signalling and its disruption

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