Our siRNA knockdown guarantee

ON-TARGETplus 2.0 siRNA reagents (SMARTpool siRNA and three out of four individual siRNAs) are guaranteed to silence target gene expression by at least 75% at the mRNA level compared to non-targeting controls as measured by qPCR when demonstrated to have been used under optimal delivery conditions (confirmed using functionally validated positive control and measured at 24 to 72 hours after transfection using 100 nM siRNA).

Note: While we guarantee knockdown >75% at 100 nM, most ON-TARGETplus 2.0 siRNA products are highly functional at 5–25 nM working concentration. Titration is recommended to maximize on-target siRNA effect and minimize any off-target behavior. Optimal siRNA delivery conditions and working concentration are experiment specific.

Please reach out to our technical support team for any questions or for guidance on optimizing the use of your siRNA products.

Complete your knockout experiment by adding these supporting reagents to your order

ON-TARGETplus 2.0 siRNA controls

Validated positive controls and non-targeting negative controls designed for use with next-generation ON-TARGETplus 2.0 siRNAs. Featuring proprietary ON-TARGETplus synthetic modifications for superior specificity and minimized off-target effects. Available as pooled or single siRNAs.

ON-TARGETplus 2.0 control siRNAs deliver improved specificity, reduced seed-based off-targeting, and enhanced consistency across experiments. These controls are recommended for any siRNA workflow utilizing ON-TARGETplus 2.0 reagents. Pooled siRNA controls are recommended when additional off-target reduction is desired and are ideal for use alongside ON-TARGETplus 2.0 pooled siRNA reagents.

Select a relevant positive control to efficiently silence a robustly expressed housekeeping gene in your experimental system for confidence in your reagent’s function.

Transfection reagents

DharmaFECT transfection reagents are optimized for improved delivery and reduced toxicity. The optimal DharmaFECT reagent for your experiment will depend on your cell type. consult our DharmaFECT cell type guide for recommendations on selecting the right transfection reagent.

Order quantity guidelines

ON-TARGETplus 2.0 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 2.0 siRNAs are guaranteed to knock down with three out of four siRNA designs and SMARTpool when used at 100 nM.

ON-TARGETplus 2.0 and siRNA SMARTpool supporting data :

ON-TARGETplus and ON-TARGETplus 2.0 siRNAs potently deplete TP53 mRNA expression with minimal impact on global gene expression

U2OS cells were transfected with 25 nM ON-TARGETplus or ON-TARGETplus 2.0 siRNAs targeting the TP53 gene, or a non-targeting control (NTC) siRNA with 0.2 µL/well DharmaFECT 4 transfection reagent. A. After 48 hours, total RNA was isolated and prepped for RNAseq or RT-qPCR. A. Data plotted as a histogram showing the distribution of global transcriptional expression, represented as transcripts per million (TPM). Strong downregulation of TP53 mRNA expression was observed in all four siRNA treatment groups, denoted on the x-axis and represented as log2 transformed TPM fold change while global gene expression was largely unchanged relative to the corresponding NTC siRNA. n = 2 biologically independent samples per group. B. Relative gene expression was measured using RT-qPCR. The relative expression of TP53 was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. Robust target gene knockdown was observed with both ON-TARGETplus and ON-TARGETplus 2.0 siRNAs. n = 2 biologically independent samples per group.

ON-TARGETplus 2.0 siRNA achieves high knockdown efficiency with superior mitigation of off-targeting over competitor siRNA designs in U2OS cells

U2OS cells were transfected with 25 nM of four individual ON-TARGETplus 2.0 siRNAs and 25 nM of the ON-TARGETplus 2.0 siRNA SMARTpool along with three competitor siRNA designs targeting TP53 using vendor recommended concentrations and procedures. TP53 expression was measured via qPCR and number of unique off-targets associated with each condition assessed via RNAseq. All siRNAs achieved high knockdown efficiency, but two out of three competitor designs were associated with hundreds of unique off-targets. Additionally, the siRNA SMARTpool exhibited a superior off-target profile than three out of four individual ON-TARGETplus 2.0 siRNA designs as expected due to lower concentrations of each individual siRNA in the pool vs individual conditions.

Competitive benchmarking experiments highlight consistent potent target knockdown and minimal off-targeting using ON-TARGETplus 2.0 siRNA SMARTpools compared to competitor siRNA designs

U2OS cells were transfected with 25 nM of four individual ON-TARGETplus 2.0 siRNAs (not shown) and 25 nM of the ON-TARGETplus 2.0 siRNA SMARTpools along with three competitor siRNA designs targeting per target (BUB1, BRD2, and, DUSP1) using vendor recommended concentrations and procedures. Gene expression was measured via qPCR and number of unique off-targets associated with each condition assessed via RNAseq. In all experiments, ON-TARGETplus 2.0 siRNA SMARTpools produced potent gene knockdown with consistently lower unique off-targets compared to competitor designs.

ON-TARGETplus 2.0 siRNA delivers highly potent target gene knockdown similar to original ON-TARGETplus siRNA designs not impacted by transcriptomic shifts over time

U2OS or HCT-116 cells were transfected with 25 nM ON-TARGETplus or ON-TARGETplus 2.0 siRNAs targeting the AHCY (left) or PRDX1 (right) genes, or a non-targeting control (NTC) siRNA with 0.2 µL/well DharmaFECT 4 transfection reagent (U2OS) or DharmaFECT 2 transfection reagent (HCT-116). After 48 hours, total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. Robust target gene knockdown was observed with both ON-TARGETplus and ON-TARGETplus 2.0 siRNAs. n = 2 biologically independent samples per group.

ON-TARGETplus 2.0 siRNA mitigates newly discovered off-target effects arising from reference genome transcriptome expansion

U2OS or HEK293T cells were transfected with 25 nM ON-TARGETplus or ON-TARGETplus 2.0 siRNAs targeting the YY1AP1 gene, or a non-targeting control (NTC) siRNA with 0.2 µL/well DharmaFECT 1 transfection reagent (HEK293T) or DharmaFECT 4 transfection reagent (U2OS). After 48 hours, total RNA was isolated and prepped for RNAseq or RT-qPCR. A. Data plotted as a histogram showing the distribution of global transcriptional expression in U2OS cells, represented as transcripts per million (TPM). Strong downregulation of YY1AP1 mRNA expression was observed in all four siRNA treatment groups, denoted on the x-axis and represented as log2 transformed TPM fold change while global gene expression was largely unchanged relative to the corresponding NTC siRNA. Notably, ON-TARGETplus siRNAs also depleted expression of the off-target gene GON4L whereas ON-TARGETplus 2.0 siRNAs maintained high specificity and did not affect GON4L expression. n = 2 biologically independent samples per group. B. Relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. Robust target gene knockdown was observed with both ON-TARGETplus and ON-TARGETplus 2.0 siRNAs, however the ON-TARGETplus siRNA was found to also reduce expression of an off-target gene, GON4L. The ON-TARGETplus 2.0 siRNAs maintained high specificity and did not affect GON4L expression. n = 2 biologically independent samples per group.

Supporting Data for ON-TARGETplus siRNA dual-strand chemical modifications, the original SMARTselection algorithm, and siRNA SMARTpool format

ON-TARGETplus siRNA chemical 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 siRNA 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 siRNAs improved the false phenotype considerably, while the ON-TARGETplus SMARTpool siRNAs 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 siRNA is modified within its seed region to destabilize miRNA-like activity and improve specificity to the desired target for potent knockdown.

SMARTselection algorithmic design of siRNA 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

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

Please see our full FAQ page for all commonly asked questions about our products.

1. What is the difference between ON-TARGETplus legacy siRNA and ON-TARGETplus 2.0 siRNA?

ON-TARGETplus 2.0 siRNA is a next-generation re-design of the original ON-TARGETplus siRNA. It features improved design algorithms, incremental transcriptome realignment, and revised sequence ranking and selection to enhance specificity through minimized off-target effects and ensure consistent gene knockdown across as many transcript isoforms as possible. Legacy ON-TARGETplus designs were based on earlier transcriptome data and design selection choices reflected earlier understanding of the transcriptomic landscape.

 

2. How does ON-TARGETplus 2.0 siRNA reduce off-target effects?

Each siRNA is algorithmically designed using updated transcriptomic data and designs are curated incorporating specificity as a key variable. Seed-region selection based on low frequency endogenous seeds and the ON-TARGETplus dual-strand chemical modifications further minimize unintended interactions.

 

3. Are ON-TARGETplus 2.0 siRNAs guaranteed to knock down my target gene?

Yes. ON-TARGETplus 2.0 siRNAs are backed by a gene knockdown guarantee, ensuring consistent and dependable performance when used under recommended conditions. If our siRNA fails to deliver on this guarantee, our teams will provide free replacement of alternative designs to suit your needs.

 

4. Can I use ON-TARGETplus 2.0 siRNAs alongside my original ON-TARGETplus siRNAs and controls in an experiment?

Yes, the chemical modification pattern is the same for both products, so they are suitable to be used side-by-side in an experiment. Many original ON-TARGETplus siRNA designs are highly functional and deliver excellent ON-TARGET knockdown results.

 

5. Can ON-TARGETplus 2.0 siRNA be used in high-throughput or automated platforms?

Absolutely. ON-TARGETplus 2.0 siRNAs are available in formats compatible with automated systems, including pre-arrayed or customized plates suitable for high-throughput applications.

 

6. Are ON-TARGETplus 2.0 siRNAs available for multiple species?

Yes. Pre-designed ON-TARGETplus 2.0 siRNAs are available for human, mouse, and rat gene targets. For other species we recommend using our siDESIGN Center tool.

 

7. How does ON-TARGETplus 2.0 siRNA support genome-wide and gene-family studies?

ON-TARGETplus 2.0 collections cover whole-genome libraries as well as focused sets for commonly studied gene families, enabling both broad and targeted screening approaches. In addition, custom libraries can be built according to your desired targets using the Cherry-pick tool.

 

8. Can ON-TARGETplus 2.0 siRNA be used alongside Edit-R™ CRISPR reagents?

Yes. ON-TARGETplus 2.0 siRNA serves as an ideal orthogonal method to validate CRISPR screening hits or complement gene knockout studies with gene knockdown testing.

 

9. How do the four siRNAs provided in a SMARTpool siRNA format or set of 4 differ?

The four siRNAs provided per gene are selected from a pool of algorithmically designed and curated candidates based on a number of proprietary key variables prioritizing function, specificity, and the transcript annotations for a given gene. Each set or pool of four siRNAs is selected to contain unique seeds and, wherever possible, to target the maximum number of 100% non-overlapping genomic coordinates for a given gene within design-space limitations.