Jedrzejuk Agata, Mibus Heiko, Serek Margrethe
Faculty of Natural Sciences, Institute for Ornamental and Woody Plant Science, University of Hannover, Herrenhauser Street 2, 30419 Hannover, Germany.
ScientificWorldJournal. 2012;2012:609597. doi: 10.1100/2012/609597. Epub 2012 May 1.
In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant β-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.
原位PCR是一种能够在单个细胞和组织中检测特定核酸序列的技术。原位PCR和原位逆转录PCR是能够提高灵敏度和通量的精湛技术，但充其量只是半定量的；因此，最好先用传统方法确定表达模式，为每个探针建立合适的条件。在植物中，原位逆转录PCR广泛用于特定基因的表达定位，包括花芽和其他器官中的MADS盒基因以及其他功能特异性基因或管家基因。当无法分离特定部分时，此方法在小器官或发育早期阶段特别有用。在本文中，我们在杂交茶香月季的花芽和叶片中使用原位逆转录PCR比较了三种不同的标记和免疫检测方法。作为靶基因，我们使用了丰富的β-肌动蛋白和仅在花芽的叶片和花瓣/萼片中表达的RhFUL基因。我们使用了地高辛-11-dUTP、生物素-11-dUTP和荧光素-12-dUTP标记的核苷酸以及抗地高辛-碱性磷酸酶/链霉亲和素-荧光素标记的抗体。所有使用的方法都给出了强烈、特异的信号，并且所有这些方法都可用于玫瑰器官组织水平上的基因表达定位。
