Saha Prasenjit, Blumwald Eduardo
Department of Plant Sciences, University of California Davis, Davis, California, United States of America.
PLoS One. 2014 Aug 29;9(8):e106308. doi: 10.1371/journal.pone.0106308. eCollection 2014.
Pearl millet [Pennisetum glaucum (L.) R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.
珍珠粟[Pennisetum glaucum (L.) R.Br.]是黍亚科粮食作物和生物能源草的近亲，为开展与C4光合作用和非生物胁迫耐受性相关的功能基因组学研究提供了理想的系统。定量实时逆转录聚合酶链反应（qRT-PCR）为进行此类基因表达分析提供了一个灵敏的平台。然而，在珍珠粟的qRT-PCR分析中，缺乏用于准确转录本标准化的合适内参基因是主要限制因素。在此，我们对来自三个基因型的234个样本中的18个内参基因进行了全面评估，这些样本包括一系列不同的发育组织、激素处理和非生物胁迫条件，以确定用于qRT-PCR数据准确标准化的合适内参基因。使用稳定性指数、BestKeeper、ΔCt、Normfinder、geNorm和RefFinder程序对Ct值进行分析，结果表明PP2A、TIP41、UBC2、UBQ5和ACT是在不同实验条件下用于准确转录本标准化的最可靠内参基因。此外，我们验证了这些基因用于精确定量相对基因表达的特异性，并提供证据表明需要组合最佳内参基因才能在珍珠粟中获得内源性基因和转基因的最佳表达模式。
