Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-637 Poznań, Poland.
FEMS Yeast Res. 2020 Dec 2;20(7). doi: 10.1093/femsyr/foaa059.
Accurate quantitation of gene expression levels require sensitive, precise and reproducible measurements of specific transcripts. Normalization to a reference gene is the most common practice to minimize the impact of the uncontrolled variation. The fundamental prerequisite for an accurate reference gene is to be stably expressed amongst all the samples included in the analysis. In the present study we aimed to assess the expression level and stability of a panel of 21 genes in Yarrowia lipolytica throughout varying conditions, covering composition of the culturing medium, growth phase and strain-wild type and recombinant burdened with heterologous protein overexpression. The panel of the selected candidate genes covered those essential for growth and maintenance of metabolism and homologs of commonly used internal references in RT-qPCR. The candidate genes expression level and stability were assessed and the data were processed using dedicated computational tools (geNorm and NormFinder). The results obtained here indicated genes unaffected by the burden of overexpression (TEF1, TPI1, UBC2, SRPN2, ALG9-like, RYL1) or by the culture medium used (ACT1, TPI1, UBC2, SEC61, ODC, CLA4, FKS1, TPS1), as well as those the least (SSDH, ODC, GPD) and the most (SEC62, TPI1, IPP1) suitable for normalization of RT-qPCR data in Y. lipolytica.
准确定量基因表达水平需要对特定转录本进行敏感、精确和可重复的测量。将基因表达标准化到参考基因是最小化不可控变异影响的最常见做法。准确参考基因的基本前提是在分析中包含的所有样本中稳定表达。在本研究中，我们旨在评估 21 个基因在不同条件下的表达水平和稳定性，涵盖培养基组成、生长阶段和野生型菌株以及重组菌株中异源蛋白过表达的负担。所选候选基因的面板涵盖了那些对酵母生长和代谢维持至关重要的基因，以及 RT-qPCR 中常用的内部参考基因的同源物。使用专用计算工具（geNorm 和 NormFinder）评估候选基因的表达水平和稳定性，并处理数据。这里获得的结果表明，不受过表达负担影响的基因（TEF1、TPI1、UBC2、SRPN2、ALG9-like、RYL1）或受培养基使用影响的基因（ACT1、TPI1、UBC2、SEC61、ODC、CLA4、FKS1、TPS1），以及那些最不适合（SSDH、ODC、GPD）和最适合（SEC62、TPI1、IPP1）用于酵母 RT-qPCR 数据标准化的基因。
