Karlgren Anna, Carlsson Jenny, Gyllenstrand Niclas, Lagercrantz Ulf, Sundström Jens F
Department of Evolutionary Functional Genomics, Evolutionary Biology Center, Uppsala University.
J Vis Exp. 2009 Apr 17(26):1205. doi: 10.3791/1205.
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
如今可用的高通量表达分析技术为科学家们提供了大量的表达谱，但由于在分离单个组织方面存在问题，它们在组织特异性表达方面的分辨率有限。表达数据需要通过例如原位杂交等表达模式来进行确认和补充，原位杂交是一种用于定位细胞特异性mRNA表达的技术。原位杂交方法费力、耗时，并且常常需要根据物种和组织进行广泛的优化。在诸如针叶树挪威云杉（Picea abies）这样的木本物种中进行原位实验相对更困难。在这里，我们展示了一种改良的地高辛（DIG）原位杂交方案，该方案快速且适用于包括挪威云杉在内的多种植物物种。只需进行一些调整，包括改变核糖核酸酶（RNase）处理和蛋白酶K浓度，我们就可以使用该方案来研究一种裸子植物和两种被子植物物种（挪威云杉、拟南芥和甘蓝型油菜）雄性生殖器官中同源基因的组织特异性表达。该方案对所研究的物种和基因同样有效。在拟南芥和甘蓝型油菜的第二轮和第三轮花器官中观察到了AtAP3和BnAP3，在挪威云杉雄球果的小孢子叶中观察到了DAL13。对于挪威云杉，用于使组织通透的蛋白酶K浓度必须提高到3 mg/ml而不是1 mg/ml，这可能是由于组织更紧密以及酚类和多糖水平更高。对于所有物种，由于信号强度降低且特异性没有相应提高，因此取消了RNase处理。通过比较开花植物和针叶树同源基因的组织特异性表达模式，我们证明了这里展示的DIG原位方案只需进行微小调整，就可以应用于多种植物物种。因此，该方案避免了广泛的物种特异性优化以及费力地使用放射性标记探针，转而采用地高辛标记探针。我们选择在我们的影片中展示该方案中技术要求较高的步骤。
