Reference Gene Validation for Quantitative Real-time PCR Studies in Amphibian Kidney-derived A6 Epithelial Cells

Abstract

Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.

Reference gene screening of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans for quantitative real-time PCR studies

“…All too often, only one reference gene or even non-validated reference genes are used in an RT-qPCR setup which can lead to misleading results 18 . A careful selection of thoroughly validated reference genes prior to performing qPCR experiments is therefore recommended.…”

Hypothesis-driven dragging of transcriptomic data to analyze proven targeted pathways in Rhinella arenarum larvae exposed to organophosphorus pesticides

“…In a comprehensive study conducted with X. laevis , rl8 and g3pdh were stable as reference genes during the first embryonic stages, odc1 was stable in the initial and final stages, and h4 was adequate throughout embryonic development 27 . In another study in X. laevis , e- ef1a1 and sub1 were identified as the genes whose expression remained more stable, which would allow their use as reference genes 28 . From a list of 9 genes fulfilling the requirements as HK genes in R. arenarum , we successfully tried actb and rl8 in qPCR assays, suggesting that these are good candidates for future qPCR analyses in different environmental studies involving this native species.…”

Pushing the Partnership Forward

“…Finally, a Belgian group from Ghent University addresses the importance of choosing correctly optimised reference genes when using the quantitative real-time polymerase chain reaction to look at gene expression levels in in vitro -treated cells. 3 This is particularly relevant to the amphibian kidney-derived epithelial cells used in their study, and they propose that these cells could replace in vivo studies on the African clawed frog ( Xenopus laevis ) animal model or, at the very least, they could be used as a prescreening tool to reduce the overall number of animals used.…”