Steinacher Claudia, Rieder Dietmar, Turner Jasmin E, Solanky Nita, Nishio Shin-Ya, Usami Shin-Ichi, Hausott Barbara, Schrott-Fischer Anneliese, Dudas Jozsef
Department of Otorhinolaryngology, Medical University Innsbruck, 6020 Innsbruck, Austria.
Institute of Bioinformatics, Medical University Innsbruck, 6020 Innsbruck, Austria.
Int J Mol Sci. 2024 Mar 2;25(5):2907. doi: 10.3390/ijms25052907.
A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (, , and ). The selected reference genes were then used to examine the effect on the expression outcome of target genes ( and ), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of and , which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.
全面的基因表达研究需要高质量的RNA提取，提取量要足以用于实时定量聚合酶链反应和下一代测序。在这项工作中，我们比较了不同的RNA提取方法，并评估了用于胎儿人内耳基因表达研究的不同参考基因。我们将从福尔马林固定石蜡包埋组织中提取的RNA与保存在-80°C的RNAlater溶液中的新鲜组织提取的RNA进行了比较，并验证了12个参考基因（从妊娠第11周到第19周）的表达稳定性。与石蜡包埋组织相比，RNAlater中新鲜组织提取的RNA产量更高，质量更好。参考基因评估显示有四个稳定表达的参考基因（、、和）。然后使用选定的参考基因来检测对已知在内耳发育过程中受到调控的靶基因（和）表达结果的影响。选定的参考基因在和的表达谱中没有显示出差异，这通过免疫染色得到了证实。结果强调了基因表达研究中RNA提取方法和参考基因选择的重要性。
