Besleaga Mihail, Vignolle Gabriel A, Kopp Julian, Spadiut Oliver, Mach Robert L, Mach-Aigner Astrid R, Zimmermann Christian
Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorfer Strasse 1a, 1060, Wien, Austria.
Center for Health and Bioresources, Competence Unit Molecular Diagnostics, AIT Austrian Institute of Technology GmbH, 1210, Vienna, Austria.
Fungal Biol Biotechnol. 2023 Mar 29;10(1):7. doi: 10.1186/s40694-023-00154-1.
The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools.
We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays.
The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.
毕赤酵母（Komagataella phaffii，以前称为巴斯德毕赤酵母Pichia pastoris）常用于异源蛋白表达，被认为是酵母的模式生物。尽管其具有重要性和应用潜力，但迄今为止，尚未评估用于通过RT-qPCR分析进行转录本分析的参考基因。在本研究中，我们在公开可用的RNA测序数据中搜索稳定表达的基因，以寻找用于毕赤酵母中通过RT-qPCR进行相对转录本分析的潜在参考基因。为了评估这些基因的适用性，我们使用了来自三种不同菌株的多种样本以及广泛的培养条件。使用常用的生物信息学工具测量并比较了9个基因的转录水平。
我们可以证明常用的参考基因ACT1表达不稳定，并鉴定出两个转录水平波动极低的基因。因此，我们建议在未来的RT-qPCR分析中，将RSC1和TAF10这两个基因同时用作毕赤酵母中通过RT-qPCR进行转录本分析的参考基因。
在RT-qPCR分析中使用ACT1作为参考基因可能会因其转录水平的不稳定性而导致结果失真。在本研究中，我们评估了多个基因的转录水平，发现RSC1和TAF10极其稳定。使用这些基因有望获得可靠的RT-qPCR结果。
