Department of Genetics, Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia.
PLoS One. 2011 May 12;6(5):e19434. doi: 10.1371/journal.pone.0019434.
Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications--geNorm, NormFinder and BestKeeper--were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.
实时荧光定量 PCR（qRT-PCR）是最精确和最广泛使用的基因表达分析方法之一。准确可靠数据的必要前提是准确选择参考基因。为了在这种经济上重要的作物中找到基因表达分析的最佳参考，我们研究了普通荞麦（Fagopyrum esculentum）中潜在参考基因的表达稳定性。最近测序的荞麦花转录组被用作序列信息的来源。在不同的植物结构（发育的两个阶段的叶片和花序以及果实）中评估了 8 个候选参考基因的表达稳定性。这些基因是在全基因组表达稳定性调查基因中鉴定为稳定的拟南芥基因和传统上使用的管家基因 GAPDH 的同源物。使用三个软件应用程序--geNorm、NormFinder 和 BestKeeper--来估计表达稳定性，得到了一致的结果。AT4G33380（未知功能表达蛋白，Expressed1）、AT2G28390（SAND 家族蛋白，SAND）和 AT5G46630（网格蛋白衔接蛋白家族蛋白，CACS）的同源物被证明是最稳定的。我们建议在使用 qRT-PCR 进行荞麦研究时，使用 Expressed1、SAND 和 CACS 组合来归一化基因表达数据。这些基因在拟南芥中是表达最稳定的五个基因之一，这强调了在其他物种中使用模式植物研究作为框架的实用性。
