A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens

Abstract

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.

Silencing of PDK1 Gene Expression by RNA Interference Suppresses Growth of Esophageal Cancer

“…Therefore, the application of PDK1 targeted RNAi to interfere or block the excessively activated P13K/Akt signal transduction pathway has been widely used in gene therapies for many malignant tumors. PDK1 siRNA effectively silences the PDK1 mRNA expression (Hsieh et al, 2004). PKD1 siRNA down-regulates the PDK1 expression specifically in breast cancer MDA-MB-231 cells, and inhibits the cell invasion capacity obviously (Min et al, 2009).…”

RNA interference and potential therapeutic applications of short interfering RNAs

“…Small-scale efforts have identified that genome-wide siRNA libraries can be prepared by various methods including chemical synthesis or enzymatic digestion of long dsRNAs. 45,57 Similarly, small-scale proof-of-principle experiments indicated that libraries can be produced by constructing shRNA expression vectors that target each gene. 45,58 Recently, two groups have prepared resources for large-scale RNAi-based screens in mammals.…”

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

“…We therefore set out to use in situ-synthesized sequences to build shRNA expression libraries targeting nearly every identified and predicted gene in the genomes of several species, including human, mouse and rat. Similar libraries have previously been constructed using conventional oligonucleotides or natural nucleic acids as starting material [28][29][30][31][32][33] . To maximize recovery of accurate clones from our highly structured templates, we used thermostable polymerases that have proofreading capability and are able to effect strand displacement.…”