Our siRNA knockdown guarantee

ON-TARGETplus siRNA reagents (SMARTpool and three of four individual siRNAs) are guaranteed to silence target gene expression by at least 75% at the mRNA level when demonstrated to have been used under optimal delivery conditions (confirmed using validated positive control and measured at the mRNA level 24 to 48 hours after transfection using 100 nM siRNA).

Note: Most ON-TARGETplus siRNA products are highly functional at 5 to 25 nM working concentration.

Validated positive controls and non-targeting negative controls utilizing patented modifications for optimal specificity. Available as pools or single siRNAs. On-TARGETplus Control siRNAs and pools are produced with the patented ON-TARGETplus modification pattern for optimal specificity. They are recommended as controls for any siRNA experiment due to their minimized off-target effects.

Our panel of Non-targeting controls permits assessment of potential non-specific effects, to find the optimal negative control for your cell type, and your assay. Pooled siRNA controls are recommended for further reduction of off-targets, and for use with SMARTpool siRNA reagents.

Select a species-specific positive control to silence an expressed housekeeping gene in your experimental cell type.

Order quantity guidelines

ON-TARGETplus reagents are routinely used at 5 to 25 nM concentration. The calculations below, based on 25 nM, are for estimation purposes only and assume no loss from pipetting.

ON-TARGETplus modifications reduce the overall number of off-targets and pooling reduces them even further

Panels (A) and (B) are representative examples of off-target signatures with and without application of ON-TARGETplus modifications to (A) a single siRNA and (B) a SMARTpool reagent. Green bars indicate genes with 2-fold or more reduction of expression when treated with the indicated siRNA reagent.The ON-TARGETplus modifications reduced the off-targets when compared to unmodified siRNA. Pooling of siRNA and the ON-TARGETplus modification pattern independently, and in combination, provide significant reduction in off-target gene silencing. Panel (C) represents quantitation of off-targets (down-regulated by 2-fold or more) induced by the indicated siRNA reagents targeting 10 different genes (4 siRNAs per gene or a single SMARTpool reagent). Off-targets were quantified using microarray analysis (Agilent) then compiled. Each shaded box represents the middle 50% of the data set. Horizontal line in box: Median value of the data set. Vertical bars: minimum and maximum data values.

False phenotypes due to off-targets are alleviated by ON-TARGETplus SMARTpool reagents while target gene knockdown is maintained

The effect of silencing ARPC1B on cell migration was studied in a breast cancer cell line. A monolayer of cells was uniformly scraped and the rate of cell migration to close the scrape (wound healing) was evaluated. Both unmodified and ON-TARGETplus siRNA reagents induced potent target knockdown. Inconsistent phenotypes due to off-target effects (red outline), were observed for cells transfected with unmodified individual siRNAs. The unmodified SMARTpool improved the false phenotype considerably, while the ON-TARGETplus SMARTpool significantly reduced off-target effects to produce a consistent phenotype.

In collaboration with Kaylene Simpson, Laura Selfors, and Joan Brugge, Harvard Medical School.

Only the ON-TARGETplus modification pattern addresses both siRNA strands for premium silencing

The ON-TARGETplus dual-strand chemical modification begins with the sense (passenger) strand being blocked from RISC uptake to favor antisense (guide) strand loading and reduce passenger strand-induced off-targets. However, the majority of siRNA off-targets are driven by the seed region of the guide strand. ON-TARGETplus is modified within its seed region to destabilize miRNA-like activity and improve specificity to the desired target for potent knockdown.

siRNA designs with low-frequency seed regions ensure fewer off-targets

ON-TARGETplus siRNA designs leverage sophisticated bioinformatics to reduce the likelihood of miRNA-like off-targets from high-frequency or highly conserved miRNA seed regions. siRNAs with low seed frequency have a significantly lower number of off-targets than siRNAs with medium or high frequency seeds. Five siRNAs with low, medium, or high frequency seed regions were transfected into HeLa cells and their associated off-target signatures assessed via global expression profiling (Agilent 22K platform). siRNA sequences were constant at positions 1 and 8-19, only the seed regions (positions 2-7) were altered.

Low frequency seeds: < 350 occurrences in the HeLa transcriptome

Medium frequency: 2500-2800 occurrences

High frequency: >3800 occurrences

ON-TARGETplus siRNA dual-strand modification pattern reduces off-targets

A 2006 publication demonstrates that off-target effects are primarily driven by antisense strand seed activity†. Therefore, sense strand inactivation alone does not decrease the total number of off-target genes.

ON-TARGETplus modifications account for both strands:

• Sense strand is modified to prevent interaction with RISC and favor antisense strand uptake
• Antisense strand seed region is modified to minimize seed-related off-targeting

The ON-TARGETplus modification pattern dramatically reduces off-targets. Off-target effects induced by the indicated siRNAs were quantified using microarray analysis. For each target, three different siRNAs were used: unmodified, sense strand-inactivated, and ON-TARGETplus-modified. Data shown represents genes down-regulated by two-fold or more. HEK293 cells were transfected with 100 nM siRNA using 0.2 μL of DharmaFECT 1. Data was analyzed at 24 hours.

1. A. L. Jackson et al., Position-specific chemical modification increases specificity of siRNA-mediated gene silencing. RNA. 12(7), 1197-1205 (July 2006).


2. A. Birmingham et al., 3'-UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods. 3(3), 199-204 (March 2006).


3. E. M. Andersonet al., Experimental validation of the importance of seed complement frequency to siRNA specificity. RNA. 14(5), 853-861 (May 2008).