ABSTRACT
Transfection of chemically synthesized short interfering RNAs (siRNAs) enables a high level of sequence-specific gene silencing. Although siRNA design algorithms have been improved in recent years, it is still necessary to prove the functionality of a given siRNA experimentally. We have functionally tested several thousand siRNAs for target genes from various gene families including kinases, phosphatases, and cancer-related genes (e.g., genes involved in apoptosis and the cell cycle). Some targets were difficult to silence above a threshold of 70% knockdown. By working with one design algorithm and a standardized validation procedure, we discovered that the level of silencing achieved was not exclusively dependent on the siRNA sequences. Here we present data showing that neither the gene expression level nor the cellular environment has a direct impact on the knockdown which can be achieved for a given target. Modifications of the experimental setting have been investigated with the aim of improving knockdown efficiencies for siRNA-target combinations that show only moderate knockdown. Use of higher siRNA concentrations did not change the overall performance of the siRNA-target combinations analyzed. Optimal knockdown at the mRNA level was usually reached 48-72 hours after transfection. Target gene-specific characteristics such as the accessibility of the corresponding target sequences to the RNAi machinery appear to have a significant influence on the knockdown observed, making certain targets easy or difficult to knock down using siRNA.
