Combinatorial RNAi for quantitative protein network analysis
Combinatorial RNAi for quantitative protein network analysis
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DOI：
10.1073/pnas.0606827104
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发表时间：
2007-04-17
影响因子：
11.1
通讯作者：
Arlt, Dorit
中科院分区：
文献类型：
--
作者：
Sahin, Ozgur;Loebke, Christian;Arlt, Dorit
阐明交叉信号通路之间的串扰事件是生物学研究中的主要挑战之一。由不同途径组成的蛋白质网络非常复杂，需要新的策略和技术来揭示相关的相互关系。在这里，我们建立了系统单、双和三重敲低的组合RNAi策略，并分别通过定量实时PCR和反相蛋白阵列定量测量残留的mRNA和蛋白质，作为数据分析的先决条件。我们的结果表明，至少三个不同基因的平行敲低是可行的，同时保持非靶向沉默和低细胞毒性。通过研究酪氨酸激酶受体 ErbB2 及其下游靶标 Akt-1 和 MEK1 在细胞侵袭中的相互作用，对该技术进行了验证。这种实验方法将多基因敲低与随后对蛋白质表达减少的定量验证相结合，是系统生物学信号通路分析的重大进步。
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.