Sahin Ozgür, Löbke Christian, Korf Ulrike, Appelhans Heribert, Sültmann Holger, Poustka Annemarie, Wiemann Stefan, Arlt Dorit
Division of Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6579-84. doi: 10.1073/pnas.0606827104. Epub 2007 Apr 9.
The elucidation of cross-talk events between intersecting signaling pathways is one main challenge in biological research. The complexity of protein networks, composed of different pathways, requires novel strategies and techniques to reveal relevant interrelations. Here, we established a combinatorial RNAi strategy for systematic single, double, and triple knockdown, and we measured the residual mRNAs and proteins quantitatively by quantitative real-time PCR and reverse-phase protein arrays, respectively, as a prerequisite for data analysis. Our results show that the parallel knockdown of at least three different genes is feasible while keeping both untargeted silencing and cytotoxicity low. The technique was validated by investigating the interplay of tyrosine kinase receptor ErbB2 and its downstream targets Akt-1 and MEK1 in cell invasion. This experimental approach combines multiple gene knockdown with a subsequent quantitative validation of reduced protein expression and is a major advancement toward the analysis of signaling pathways in systems biology.
阐明交叉信号通路之间的相互作用事件是生物学研究中的一项主要挑战。由不同通路组成的蛋白质网络的复杂性，需要新的策略和技术来揭示相关的相互关系。在这里，我们建立了一种用于系统地进行单基因、双基因和三基因敲低的组合RNA干扰策略，并且我们分别通过定量实时PCR和反相蛋白质阵列对残留的mRNA和蛋白质进行定量测量，作为数据分析的前提条件。我们的结果表明，在保持非靶向沉默和细胞毒性较低的同时，平行敲低至少三个不同基因是可行的。通过研究酪氨酸激酶受体ErbB2及其下游靶点Akt-1和MEK1在细胞侵袭中的相互作用，验证了该技术。这种实验方法将多个基因敲低与随后对蛋白质表达降低的定量验证相结合，是系统生物学中信号通路分析的一项重大进展。
