Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.
Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.
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DOI：
10.1016/j.jbiotec.2013.08.008
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发表时间：
2013-10
影响因子：
4.1
通讯作者：
Kun Zhang;Shaofang Niu;D. Di;Lindan Shi;Deshui Liu;Xiuling Cao;H. Miao;Xianbing Wang;Chenggui Han;Jialin Yu;Dawei Li;Yongliang Zhang
中科院分区：
文献类型：
--
作者：
Kun Zhang;Shaofang Niu;D. Di;Lindan Shi;Deshui Liu;Xiuling Cao;H. Miao;Xianbing Wang;Chenggui Han;Jialin Yu;Dawei Li;Yongliang Zhang
无论是全基因组的mRNA表达谱的转录组学调查和病毒诱导的基因沉默在病毒-植物互作过程中的靶基因的分子研究涉及的转录丰度的精确估计。定量实时PCR（qPCR）是mRNA定量最广泛采用的技术。为了获得可靠的定量的成绩单，最好的参考基因的识别形成的基础上的初步工作。然而，病毒感染的单子叶植物的内部控制的稳定性需要充分探讨。本研究以大麦条纹花叶病毒（Barley stripe mosaic virus，BSMV）、雀麦花叶病毒（Brome mosaic virus，BMV）、水稻黑条矮缩病毒（Rice black-streaked dwarf virus，RBSDV）和甘蔗花叶病毒（Sugarcane mosaic virus，SCMV）感染的5种单子叶植物（Brachypodium、大麦、高粱、小麦和玉米）为材料，研究了10个管家基因（ACT、EF 1 α、FBOX、GAPDH、GTPB、PP 2A、SAND、TUBβ、UBC 18和UK）作为qPCR参考基因的适用性。通过使用三种不同的算法，最合适的参考基因或它们的组合被确定为不同的实验集，其有效性的表达研究的标准化进一步验证了一个研究良好的PR-1基因的定量分析。这些结果有利于选择理想的参考基因，更准确的基因表达研究在病毒感染的单子叶植物。
Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus–plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT,EF1α,FBOX,GAPDH,GTPB,PP2A,SAND,TUBβ,UBC18andUK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses includingBarley stripe mosaic virus(BSMV),Brome mosaic virus(BMV),Rice black-streaked dwarf virus(RBSDV) andSugarcane mosaic virus(SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studiedPR-1gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots.