A comparison of target gene silencing at 5 and 100 nM concentration illustrates the high potency of siGENOME SMARTpool reagents. 5 or 100 nM siGENOME SMARTpool siRNA reagents targeting the indicated genes was transfected into HeLa cells (10K cells/well) using 0.1 μL/well DharmaFECT 1. The negative control (NTC pool) was siGENOME Non-Targeting Pool #2.
Unmodified and sense strand-inactivated siRNAs were used to target five genes. In four cases, off-targets were increased due to enhanced RISC loading of the antisense strand when the sense strand was modified. The unmodified siRNAs have natural guide-strand loading characteristics. All siRNAs had comparable silencing potency. Data shown represents genes down-regulated by twofold or more. HEK293 cells were transfected with 100 nM siRNA using 0.2 μL of DharmaFECT 1. Data was analyzed at 24 hours by genomewide microarray analysis (Agilent).
Why not modify ALL siGENOME siRNAs to ensure proper strand loading? It has been demonstrated by Dharmacon scientists1 and others2 that forcing antisense (guide) strand entry into RISC may actually INCREASE off-targets due to increased loading of the guide strand and resulting off-target activity by its seed region. siGENOME siRNAs are designed with thermodynamic properties to naturally facilitate guide strand entry to RISC, which has been demonstrated to correlate with functionality1. However, in cases where a high-scoring siGENOME siRNA does not possess ideal strand-loading characteristics, a sense (passenger) strand-inhibiting chemical modification (ON-TARGET) is utilized to promote guide strand entry. Approximately 20% of siGENOME siRNAs carry the ON-TARGET modification.
Target gene silencing is maintained while off-target signatures are reduced with a SMARTpool siRNA reagent when compared to its four constituent siRNAs.
Total RNA for microarray extracted from HeLa cells 24 hours post-transfection. Green indicates mRNA reduced by two-fold or more (Agilent 22K Platform).