A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens.
Gene silencing through RNA interference (RNAi) has been established as a means of conducting reverse genetic studies. In order to better understand the determinants of short interfering RNA (siRNA) knockdown for use in high-throughput cell-based screens, 148 siRNA duplexes targeting 30 genes within the PI3K pathway were selected and synthesized. The extent of RNA knockdown was measured for 22 genes by quantitative real-time PCR. Analysis of the parameters correlating with effective knockdown showed that (i) duplexes targeting the middle of the coding sequence silenced significantly poorer, (ii) silencing by duplexes targeting the 3'UTR was comparable with duplexes targeting the coding sequence, (iii) pooling of four or five duplexes per gene was remarkably efficient in knocking down gene expression and (iv) among duplexes that achieved a >70% knockdown of the mRNA there were strong nucleotide preferences at specific positions, most notably positions 11 (G or C) and 19 (T) of the siRNA duplex. Finally, in a proof-of-principle pathway-wide cell-based genetic screen, conducted to detect negative genetic regulators of Akt S473 phosphorylation, both known negative regulators of this phosphorylation, PTEN and PDK1, were found. These data help to lay the foundation for genome-wide siRNA screens in mammalian cells.
Pubmed ID: 14769947 RIS Download
Cell Line | Gene Targeting | Humans | Phenotype | Phosphatidylinositol 3-Kinases | Phosphorylation | Polymerase Chain Reaction | Protein-Serine-Threonine Kinases | Proteins | Proto-Oncogene Proteins | Proto-Oncogene Proteins c-akt | RNA Interference | RNA, Messenger | RNA, Small Interfering | Signal Transduction