RNA-seq-based selection of reference genes for RT-qPCR analysis of pitaya
RNA-seq-based selection of reference genes for RT-qPCR analysis of pitaya
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基于RNA-seq的火龙果RT-qPCR分析内参基因选择
DOI：
10.1002/2211-5463.12678
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发表时间：
2019-07-11
期刊：
影响因子：
2.6
通讯作者：
Xia, Kuaifei
中科院分区：
文献类型：
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作者：
Nong, Quandong;Yang, Yongchao;Xia, Kuaifei
逆转录定量实时PCR（RT-qPCR）是测量基因表达水平的主要工具，选择合适的内参基因对于在各种实验条件下获得准确且可重复的基因表达结果至关重要。然而，尚未对火龙果（Hylocereus undatus Britt.）的参考基因进行系统评价。在这里，我们检测了五个候选参考基因，即延伸因子 1-α (HuEF1-α)、18S 核糖体 RNA (Hu18S rRNA)、泛素 (HuUBQ)、肌动蛋白 (HuACT) 和泛素结合酶 (HuUQT) 在火龙果中不同条件下的表达。使用两个计算程序评估这五个基因的表达稳定性：geNorm 和NormFinder。通过标准化磷酸甘油酸激酶（HuPGK）和乙烯反应转录因子（HuERF）基因的表达，进一步验证了结果。我们的结果表明，在所有检查的实验条件下，联合使用 HuUBQ 和 HuUQT 是最稳定的参考。 HuEF1-alpha、HuUBQ 和 HuUQT 是盐胁迫、干旱胁迫和热胁迫下以及不同品种中最稳定的前三个参考基因。 HuEF1-α、HuACT 和 HuUQT 在不同组织中表现出最稳定的表达模式。我们的结果将使研究人员能够选择最合适的参考基因，用于不同条件下火龙果的基因表达研究。
Reverse-transcription quantitative real-time PCR (RT-qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation of reference genes in pitaya (Hylocereus undatus Britt.) has been performed. Here, we examined the expression of five candidate reference genes, namely elongation factor 1-alpha (HuEF1-alpha), 18S ribosomal RNA (Hu18S rRNA), ubiquitin (HuUBQ), actin (HuACT), and ubiquitin-conjugating enzyme (HuUQT), under different conditions in pitaya. The expression stabilities of these five genes were evaluated using two computation programs: geNorm and NormFinder. The results were further validated by normalizing the expression of the phosphoglycerate kinase (HuPGK) and ethylene-responsive transcription factor (HuERF) genes. Our results indicate that combined use of HuUBQ and HuUQT is the most stable reference under all of the experimental conditions examined. HuEF1-alpha, HuUBQ, and HuUQT are the top three most stable reference genes under salt stress, drought stress, and heat stress, and across different cultivars. HuEF1-alpha, HuACT, and HuUQT exhibited the most stable expression patterns across different tissues. Our results will allow researchers to select the most appropriate reference genes for gene expression studies of pitaya under different conditions.