Protein Levels and mRNA Knockdown: The importance of accurate protein quantification in RNA silencing using PCR, Western Blotting and In-Cell Western™ Assays
Recent Poster Presentation: A collaboration of Gregory R. Hoffman of Harvard University School of Medicine and Sally Weldon, LI-COR Biosciences, was presented as a poster at the recent Society for Biomolecular Science meeting held October 18th and 19th in North Carolina.
Cells transfected with Akt1 siRNA or scrambled siRNA (NS1) were subjected to both quantitative real time PCR (qRT-PCR) and quantitative Western blot analysis. Results suggest that the protein turnover of Akt1 is slow relative to the kinetics of mRNA knockdown and highlight the importance of accurately quantifying protein levels in siRNA experiments. The poster also shows the application of the In Cell Western assay to the development of a high throughput screening assay to monitor changes in ribosomal protein S6 (rpS6) phosphorylation in response to siRNA knockdown of components of the mTORC1 signaling network.