Combined Use of RNAi and Quantitative Proteomics to Study Gene Function in Drosophila

Molecular Cell, 2008, 31, 762-72 published on 05.09.2008
Molecular Cell, online article

RNA interference is a powerful way to study gene function and is frequently combined with microarray analysis. Here we introduce a similar technology at the protein level by simultaneously applying Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and RNA interference (RNAi) to Drosophila SL2 cells. After knockdown of ISWI, an ATP-hydrolyzing motor of different chromatin remodeling complexes, we obtained a quantitative proteome of more than 4,000 proteins. ISWI itself was reduced 10-fold as quantified by SILAC. Several hundred proteins were significantly regulated and clustered into distinct functional categories. Acf-1, a direct interaction partner of ISWI, is severely depleted at the protein, but not the transcript, level; this most likely results from reduced protein stability. We found little overall correlation between changes in the transcriptome and proteome with many protein changes unaccompanied by message changes. However, correlation was high for those mRNAs that changed significantly by microarray.

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