Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, 110021, India.
Interdisciplinary Centre for Plant Genomics, University of Delhi South Campus, New Delhi, 110021, India.
Sci Rep. 2018 Feb 16;8(1):3203. doi: 10.1038/s41598-018-21374-1.
The circadian clock in plants is the intrinsic rhythmic expression of thousands of genes in a 24 h period, which is set by the day-night cycles in the environment. The study of the circadian clock often requires expression profiling of genes over a large number of samples for which RT-qPCR is invariably used. Reliability of the results depends largely on the house-keeping genes, which serve as control and thus should be chosen carefully to prevent erroneous results. In this study, ten house-keeping genes were chosen from rice for stability analysis with 48 tissue samples harvested from plants subjected to diurnal/circadian cycles. Although, all the genes were found to be stable, however, six of them showed cyclic expression patterns and caused major changes in the expression profiles of the target genes when used to normalize their expression data, thereby making them poor candidates for diurnal/circadian studies. In conclusion, reference genes need to be selected for diurnal/circadian studies with extra caution as more than 80% of transcriptome in plants undergoes cycling, which remains undetected by the gene stability assessment software and can severely affect the RT-qPCR based gene expression profiling. The geometric mean of two or more most stable reference genes is hence recommended for diurnal/circadian studies in plants.
植物中的生物钟是在 24 小时周期内，数千个基因内在有节奏表达的结果，这是由环境中的昼夜循环设定的。生物钟的研究通常需要对大量样本中的基因进行表达谱分析，而实时定量 PCR（RT-qPCR）则是常用的方法。结果的可靠性在很大程度上取决于内参基因，内参基因作为对照，因此应谨慎选择，以防止出现错误的结果。在这项研究中，从水稻中选择了 10 个看家基因进行稳定性分析，共采集了 48 个组织样本，这些样本来自经历昼夜/生物钟周期的植物。尽管所有基因都被发现是稳定的，但其中 6 个基因表现出周期性表达模式，当用于归一化目标基因的表达数据时，会导致目标基因的表达谱发生重大变化，从而使它们成为昼夜/生物钟研究的不理想候选基因。总之，在进行昼夜/生物钟研究时，需要格外小心地选择参考基因，因为植物中超过 80%的转录组会发生循环，而这一现象会被基因稳定性评估软件所忽略，这可能会严重影响基于 RT-qPCR 的基因表达谱分析。因此，建议在植物的昼夜/生物钟研究中使用两个或更多最稳定的参考基因的几何平均值。
