Combinatorial RNAi for quantitative protein network analysis.
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.
Sahin
Ozgür
O
Division of Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
Löbke
Christian
C
Korf
Ulrike
U
Appelhans
Heribert
H
Sültmann
Holger
H
Poustka
Annemarie
A
Wiemann
Stefan
S
Arlt
Dorit
D
D045744:Cell Line, Tumor; D020650:Combinatorial Chemistry Techniques; D006801:Humans; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020239:Receptor Cross-Talk / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D049490:Systems Biology
D011506:Proteins; D034741:RNA, Small Interfering