Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.
Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.
复制标题
DOI：
10.1186/1471-2229-10-49
复制
发表时间：
2010-03-21
影响因子：
5.3
通讯作者：
Alves-Ferreira M
中科院分区：
文献类型：
--
作者：
Artico S;Nardeli SM;Brilhante O;Grossi-de-Sa MF;Alves-Ferreira M
通过参考基因或管家基因进行标准化，可以从逆转录实时定量聚合酶链反应（qPCR）中获得更准确和可靠的结果。最近的研究表明，没有一个管家基因对所有实验都是通用的。因此，合适的参考基因应该是任何qPCR分析的第一步。在植物中对管家基因的鉴定研究较少。因此，由于缺乏合适的参考基因，对棉花等重要作物的qPCR研究受到阻碍。通过使用geNorm和Normalware实现的两种不同的算法，我们评估了棉花中9个候选参考基因的基因表达：GhACT 4，GhEF 1 α5，GhFBX 6，GhPP 2A 1，GhMZA，GhPTB，GhGAPC 2，Ghβ TUB 3和GhUBQ 14。候选参考基因在23个实验样品中进行了评估，包括6个不同的植物器官，花发育的8个阶段，果实发育的4个阶段和花轮枝。GhPP 2A 1和GhUBQ 14基因的表达在所有样品中是最稳定的，并且当检查不同的植物器官时也是如此。GhACT 4和GhUBQ 14在花发育过程中稳定表达，GhACT 4和GhFBX 6在花轮生组织中稳定表达，GhMZA和GhPTB在果实发育过程中稳定表达。我们的分析为每个实验组提供了最合适的参考基因组合，作为可靠的qPCR数据标准化的内部对照。此外，为了说明棉花参考基因的使用，我们检查了两个棉花MADS盒基因在不同植物和花器官中以及花发育期间的表达。我们已经测试了9个候选基因的表达稳定性在一组23个组织样品从棉花植物分为五个不同的实验组。作为评估的结果，我们建议使用GhUBQ 14和GhPP 2A 1管家基因作为上级参考，用于不同棉花植物器官中基因表达测量的标准化; GhACT 4和GhUBQ 14用于花发育，GhACT 4和GhFBX 6用于花器官，GhMZA和GhPTB用于果实发育。我们还提供了引物序列，其在qPCR实验中的性能得到证明。这些基因将使这种重要作物的基因表达研究的qPCR结果更加准确和可靠，这是天然纤维的主要来源，也是食用油的重要来源。使用真正的参考基因允许详细和准确的时间和空间表达模式的两个MADS-box基因在棉花的表征。
Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in different cotton plant organs; GhACT4 and GhUBQ14 for flower development, GhACT4 and GhFBX6 for the floral organs and GhMZA and GhPTB for fruit development. We also provide the primer sequences whose performance in qPCR experiments is demonstrated. These genes will enable more accurate and reliable normalization of qPCR results for gene expression studies in this important crop, the major source of natural fiber and also an important source of edible oil. The use of bona fide reference genes allowed a detailed and accurate characterization of the temporal and spatial expression pattern of two MADS-box genes in cotton.
登录
查看更多内容
影响因子：
5.9
作者：
Guo, Yulong;Zhu, Qinlong;Li, Mingyang
通讯作者：
Li, Mingyang
影响因子：
2.7
作者：
Ransbotyn, Vanessa;Reusch, Thorsten B. H.
通讯作者：
Reusch, Thorsten B. H.
影响因子：
3.1
作者：
Lightfoot, Damien J.;Malone, Katharine M.;Orford, Sharon J.
通讯作者：
Orford, Sharon J.
影响因子：
5.3
作者：
Exposito-Rodriguez, Marino;Borges, Andres A.;Borges-Perez, Andres;Perez, Jose A.
通讯作者：
Perez, Jose A.
影响因子：
--
作者：
Barsalobres-Cavallari CF;Severino FE;Maluf MP;Maia IG
通讯作者：
Maia IG