Fausto Anna Karoline Silva, Silva Tatiane da Franca, Romanel Elisson, Vaslin Maite F S
Lab. Virologia Molecular Vegetal, Depto. Virologia, IMPPG, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, Brasil.
Departamento de Biotecnologia, Escola de Engenharia de Lorena (EEL), Universidade de São Paulo (USP), Lorena, SP, Brasil.
PLoS One. 2017 Apr 17;12(4):e0174722. doi: 10.1371/journal.pone.0174722. eCollection 2017.
Cotton (Gossypium hirsutum) is the most important non-food plant in the world. Studies concerning the fiber quality and plant fitness of cotton at molecular level depend on high sensitive and reproducible gene-expression assays. However, only a few reports have described genes suitable for normalizing gene expression data. In this study, we report for the first time that microRNAs (miRNAs) are reliable reference genes (RGs) for cotton gene expression normalization in quantitative real-time reverse transcription (RT)-PCR. The stability of cotton miRNAs was assayed in root, stem, leaf and flower samples from three different cultivars [FiberMax (FM966), Delta Opal (DO) and Cedro] and under conditions of biotic stress caused by infection with Cotton leafroll dwarf virus (CLRDV). The stability of mRNAs already described as reference genes in cotton was also assessed. The geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to select the best reference genes. In 8 of the 12 sets tested, miRNAs (miR172, 168 and 390) were found to be the best RGs. To validate the best selected RGs, miR159, miR164, miR2118, miR2910, miR3476, GhDCL2 and GhDCL4 expression levels were evaluated under biotic stress conditions, and miR164 and a putative myo-inositol oxigenase gene (GhMIOX) were assessed in leaves and flowers. The RGs selected in this work proved to be excellent reference genes in the two cases studied. Our results support the use of miRNAs as reference genes for miRNA and protein-coding genes.
棉花（陆地棉）是世界上最重要的非粮食作物。在分子水平上对棉花纤维品质和植株适应性的研究依赖于高灵敏度且可重复的基因表达分析方法。然而，仅有少数报道描述了适用于标准化基因表达数据的基因。在本研究中，我们首次报道了微小RNA（miRNA）是定量实时逆转录（RT）-PCR中用于棉花基因表达标准化的可靠内参基因（RG）。我们检测了来自三个不同品种[FiberMax（FM966）、Delta Opal（DO）和Cedro]的根、茎、叶和花样本中棉花miRNA的稳定性，以及在感染棉花卷叶矮缩病毒（CLRDV）引起的生物胁迫条件下棉花miRNA的稳定性。我们还评估了已被描述为棉花内参基因的mRNA的稳定性。使用geNorm、NormFinder、BestKeeper和ΔCt算法来选择最佳内参基因。在测试的12组样本中的8组中，发现miRNA（miR172、168和390）是最佳内参基因。为了验证最佳选择的内参基因，在生物胁迫条件下评估了miR159、miR164、miR2118、miR2910、miR3476、GhDCL2和GhDCL4的表达水平，并在叶和花中评估了miR164和一个假定的肌醇氧化酶基因（GhMIOX）。在本研究中选择的内参基因在这两个研究案例中均被证明是优秀的内参基因。我们的结果支持将miRNA用作miRNA和蛋白质编码基因的内参基因。
