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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.
Figure 1. RNA-seq expression of 14 candidate reference genes and four previously used reference genes. (A) Heatmap of 18 candidate genes’ total RNA expression (TPM, transcript per million) during the 14 developmental stages of Xenopus laevis development. (B) Variation of 18 candidate genes’ total RNA expression (TPM) at the different stages as a box plot. (C) Venn diagram outlining the two different classes of top ranking genes, with oocyte and without oocyte.
Figure 2. Ct values of 16 reference genes during different developmental stages. Variation of 16 candidate genesâ RNA expression (Ct) assessed using RT-qPCR in (A) whole embryos during early developmental stages and brain during metamorphic stages, (B) whole embryos during early developmental stages and in (C) brain during metamorphic stages.
Figure 3. Stability of genes during different developmental periods. Gene expression and stability calculated using geNorm (qBase+). Different developmental series in (A) whole embryos during early developmental stages and (B) brain during metamorphic stages. The significance of the different developmental series are, A; whole embryo developmental stages including unfertilised egg (NF0âNF50), B; whole embryo developmental stages (NF1âNF50), C; whole embryo developmental stages prior to thyroid gland formation including unfertilised egg (NF0âNF41), D; whole embryo developmental stages prior to thyroid gland formation (NF1âNF41), E; whole embryo developmental stages from 1 cell to mid blastula (NF1âNF10), F; whole embryo developmental stages after gastrulation and prior to thyroid gland formation (NF21âNF37), G; whole embryo developmental stages after gastrulation and thyroid gland formation (NF21âNF41), H; whole embryo developmental stages during thyroid gland formation (NF37âNF50), AA, Brain tissue from early developmental period, metamorphosis and Juvenile (NF41âNF66), and AB; Brain tissue from metamorphosis and Juvenile stages (NF50âNF66). (C) Different series and their corresponding genes ranked using geNorm M. Highlighted genes are minimum combination of high ranking reference genes required in the series using geNorm V.
Figure 4. Comparison of four different statistical algorithms used to calculate reference gene stability. Four different statistical algorithms, geNorm, delta-CT, NormFinder and BestKeeper, were used to compare the gene expression and stability of the 16 candidate reference genes. Different developmental series (A) All samples including whole embryos and brain during metamorphosis, (B) whole embryos including unfertilised oocyte, (C) whole embryos without the unfertilised oocyte, and (D) brains during metamorphosis.
Figure 5. Ct values of 16 reference genes in NF48 brain exposed to thyroid (T3). Variation of 16 reference genes mRNA expression (Ct) assessed using RT-qPCR (A) Control vs T3 (B) Control vs T3 vs NH3 (T3 antagonist) vs Triclosan. Analysis using four different statistical algorithms, geNorm, delta-CT, NormFinder and BestKeeper. Series of different comparison of experimental conditions (C) Control vs T3 (D) Control vs T3 vs NH3 (T3 antagonist) vs Triclosan.
Figure 6. Relative fold changes of thyroid signalling genes using reference genes in NF48 X. laevis brains. Results are presented as fold changes. The previously identified two candidate reference genes, ube2m.S and ralb.S, were used to normalise the expression of the thyroid signalling genes (A) dio1, (B) dio2, (C) dio3, (C) tr
α, and (E) tr
β. Statistics used one way ANOVA. Values represent meansâ±âSD (nâ=â3); *Pâ<â0.01, and **Pâ<â0.001.
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