Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.
Department of Energy Institute of Genomics and Proteomics, University of California Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2021 Apr 28;16(4):e0233249. doi: 10.1371/journal.pone.0233249. eCollection 2021.
Quantitative gene expression analysis is an important tool in the scientist's belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter's unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.
定量基因表达分析是科学家工具包中的重要工具。无论使用传统的 RT-PCR（逆转录聚合酶链反应）还是 qRT-PCR（实时定量 PCR；qPCR），准确的定量基因表达分析都需要鉴定均匀表达的参考基因。在Stramenopiles（真核生物的主要分支，包括褐藻）中，由于缺乏可用的分子工具，这种研究中缺乏已知的参考基因。在这里，我们提出了一组 9 个参考基因（延伸因子 1 阿尔法（EF1A）、延伸因子 2 阿尔法（EF2A）、延伸因子 1 贝塔（EF1B）、14-3-3 蛋白、泛素连接酶（UBCE2）、甘油醛-3-磷酸脱氢酶（GAPDH）、肌动蛋白相关蛋白复合物（ARP2/3）、核糖体蛋白（40s；S23）和肌动蛋白），用于褐藻 Fucus distichus。这些参考基因在六种非生物胁迫条件（干燥、光照和温度改变、激素添加、污染物暴露、营养添加和创伤）下的成年孢子体上进行了测试。使用标准方法定量评估了这些基因在不同条件下作为参考基因的适用性，大多数测试基因的评价结果都是有利的。然而，我们表明，应根据条件选择归一化基因。我们建议每个实验至少使用两个参考基因，列出了这里测试条件下的推荐基因对，并提供了一种用于确定适合实验者独特设计的合适基因集的程序。随着人们对褐藻生物学兴趣的不断增加以及伴随而来的分子工具的发展，这里测试的各种实验条件使这项研究成为基础生物学和了解褐藻谱系中应激反应的宝贵资源。
