Melgar-Rojas Pedro, Alvarado Juan Carlos, Fuentes-Santamaría Verónica, Gabaldón-Ull María Cruz, Juiz José M
Instituto de Investigación en Discapacidades Neurológicas (IDINE), School of Medicine, University of Castilla-La Mancha, Campus in Albacete, Albacete, Spain.
PLoS One. 2015 Sep 14;10(9):e0138027. doi: 10.1371/journal.pone.0138027. eCollection 2015.
The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) requires adequate normalization in order to ensure accurate results. The use of reference genes is the most common method to normalize RT-qPCR assays; however, many studies have reported that the expression of frequently used reference genes is more variable than expected, depending on experimental conditions. Consequently, proper validation of the stability of reference genes is an essential step when performing new gene expression studies. Despite the fact that RT-qPCR has been widely used to elucidate molecular correlates of noise-induced hearing loss (NIHL), up to date there are no reports demonstrating validation of reference genes for the evaluation of changes in gene expression after NIHL. Therefore, in this study we evaluated the expression of some commonly used reference genes (Arbp, b-Act, b2m, CyA, Gapdh, Hprt1, Tbp, Tfrc and UbC) and examined their suitability as endogenous control genes for RT-qPCR analysis in the adult Wistar rat in response to NIHL. Four groups of rats were noise-exposed to generate permanent cochlear damage. Cochleae were collected at different time points after noise exposure and the expression level of candidate reference genes was evaluated by RT-qPCR using geNorm, NormFinder and BestKeeper software to determine expression stability. The three independent applications revealed Tbp as the most stably expressed reference gene. We also suggest a group of top-ranked reference genes that can be combined to obtain suitable reference gene pairs for the evaluation of the effects of noise on gene expression in the cochlea. These findings provide essential basis for further RT-qPCR analysis in studies of NIHL using Wistar rats as animal model.
逆转录定量聚合酶链反应（RT-qPCR）需要进行适当的标准化以确保结果准确。使用参考基因是使RT-qPCR检测标准化的最常用方法；然而，许多研究报告称，常用参考基因的表达比预期更具变异性，这取决于实验条件。因此，在进行新的基因表达研究时，对参考基因稳定性进行适当验证是必不可少的一步。尽管RT-qPCR已被广泛用于阐明噪声性听力损失（NIHL）的分子关联，但截至目前，尚无报告表明对用于评估NIHL后基因表达变化的参考基因进行了验证。因此，在本研究中，我们评估了一些常用参考基因（Arbp、b-Act、b2m、CyA、Gapdh、Hprt1、Tbp、Tfrc和UbC）的表达，并检测了它们作为成年Wistar大鼠NIHL反应中RT-qPCR分析内参基因的适用性。将四组大鼠暴露于噪声中以造成永久性耳蜗损伤。在噪声暴露后的不同时间点收集耳蜗，使用geNorm、NormFinder和BestKeeper软件通过RT-qPCR评估候选参考基因的表达水平，以确定表达稳定性。这三种独立的应用表明Tbp是表达最稳定的参考基因。我们还提出了一组排名靠前的参考基因，它们可以组合起来以获得合适的参考基因对，用于评估噪声对耳蜗基因表达的影响。这些发现为以Wistar大鼠作为动物模型的NIHL研究中进一步的RT-qPCR分析提供了重要依据。
