Department of Plant Sciences, Agrifood Research and Technology Centre of Aragon (CITA), Avd. Montañana 930, 50059 Zaragoza, Spain.
AgriFood Institute of Aragon-IA2, CITA-University of Zaragoza, 50013 Zaragoza, Spain.
Int J Mol Sci. 2023 Feb 3;24(3):3052. doi: 10.3390/ijms24033052.
Lettuce is a popular vegetable source of bioactive compounds, like anthocyanins, powerful antioxidants present in red and semi-red varieties. Selection of reliable reference genes (RGs) for the normalization of real-time quantitative PCR (qPCR) data is crucial to obtain accurate gene expression results. Among the genes with totally unrelated biological functions, six candidate RGs (, , , , , and with low variation in expression according to RNA-seq analyses, were selected for future expression studies of anthocyanin-related genes in three different experiments: leaf colour comparison (green vs. red) in commercial varieties; tissue comparison (leaf vs. stem) in a wild relative; and drought stress experiment in commercial and traditional varieties, and a wild relative. Expression profiles of the candidate RGs were obtained by qPCR and their stability was assessed by four different analytical tools, geNorm, NormFinder, BestKeeper, and Delta Ct method, all integrated in RefFinder. All results considered, we recommend to be used as RG for the leaf colour experiment and for the tissue and drought stress ones, as they were the most stable genes in each case. RNA-seq is useful to preselect novel RGs although validation by qPCR is still advisable. These results provide helpful information for gene expression studies in spp. under the described conditions.
生菜是一种富含生物活性化合物的常见蔬菜，如花色苷，这是一种存在于红色和半红色品种中的强抗氧化剂。选择可靠的内参基因（RGs）用于实时定量 PCR（qPCR）数据的标准化对于获得准确的基因表达结果至关重要。在具有完全不同生物学功能的基因中，根据 RNA-seq 分析，选择了 6 个候选 RG（、、、、和）进行未来的花色苷相关基因表达研究，这些基因在三个不同的实验中进行：商业品种的叶片颜色比较（绿色与红色）；野生近缘种的组织比较（叶片与茎）；以及商业品种和传统品种以及野生近缘种的干旱胁迫实验。通过 qPCR 获得候选 RG 的表达谱，并通过 geNorm、NormFinder、BestKeeper 和 Delta Ct 方法这四种不同的分析工具评估其稳定性，所有结果都整合在 RefFinder 中。综合所有结果，我们建议在叶片颜色实验中使用 作为 RG，在组织和干旱胁迫实验中使用 ，因为它们在每种情况下都是最稳定的基因。RNA-seq 可用于预筛选新的 RG，但仍建议通过 qPCR 进行验证。这些结果为描述条件下 种的基因表达研究提供了有用的信息。
