Optimized siRNA Pools for Reliable Gene Silencing

siPOOLs

siPOOLs are complex siRNA pools of 30 pre-defined designed siRNAs per target gene. Each siRNA is present at  picomolar concentration to ensure high specificity, minimized off‑target effects, and robust, reproducible results.

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siRNA-mediated knockdown of genes

Small interfering RNAs (siRNAs) are short, double-stranded RNA molecules that are a key player in RNA interference (RNAi). Naturally occurring siRNAs are processed by the type III Dicer ribonuclease and then loaded into the RNAi-Induced Silencing Complex (RISC) where the guide strand of the siRNA binds to the central Argonaut (AGO) protein. This complex can scan the transcriptome and recognize matching mRNAs that are cleaved through the nuclease activity of AGO, preventing translation of this gene.

By using synthetic siRNAs, we can use this powerful mechanism to effectively knock down specific target genes.

Optimized and complex siRNA design

With 30 defined siRNA sequences, siPOOLs ensure optimized cooperative targeting of all gene transcripts, including predicted isoforms.

Proprietary siRNA design algorithms select the most potent siRNAs based on thermodynamic properties that favor guide strand loading into the RISC.

Using latest RefSeq annotations and genome-wide paralogue filtering, siPOOLs are designed for maximum coverage of all targeted transcripts with high specificity.

siPOOLs offer complex siRNA pools for gene silencing

siPOOLs use the unique approach of complex siRNA pooling with 30 specifically designed siRNAs targeting different sequences within the same target gene.

This strategy achieves the following:

  • Off-target dilution by very low concentration of each siRNA
  • Robust, maximum silencing by cooperative activity of many siRNAs

 

Overcoming off-target effects of siRNAs

One major challenge when working with siRNAs is the high rate of off-target effects that stem from the 6-7 bp “seed” sequence on the guide strand. Genes that match these “seeds” can also be affected by the effects of the siRNA, creating unwanted side effects, with these effects showing up as the top hits in conventional siRNA screens.

The pooling of 30 different siRNAs in within siPOOLs dilutes the possible off-target effect of each siRNA to a minimum. Simultaneously, this approach increases the target specificity of the knockdown through cooperative targeting, increasing probabilities of highly efficient siRNAs and for the inclusion of isoforms.

As such, complex siRNA pooling enhances silencing robustness efficiently, providing maximum silencing without the need for extensive experimental validation.

Single siRNA
30 siRNAs combined - siPOOL

Proven in Research – Publications

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What Scientists Say – Testimonials

"Our lab uses arrayed, high-throughput functional genomic screening in primary neurons to identify potential neuroprotective drug targets. Having tested over 75,000 siRNA sequences, it is quite apparent that off-target effects dominate siRNA-mediated phenotypes. In contrast, in our hands, siPOOLs have much greater predictive power in that phenotypes we see with these (and we have tested approximately 15) can be reproduced using cells containing conventional knockouts for the same genes. We now routinely use siPOOLs and are moving away from single siRNAs."

Dr. Derek Welsbie (Assistant Professor, University of California San Diego, USA)

"In our group, siPOOLs by siTOOLs for specific and sustained gene knockdown are used for several years now. We have tested and worked with approx. 35-40 siPOOLs by now. siPOOLs are easy in handling, and mostly effective in transient specific gene knockdown experiments. We could not detect any side effects, and the available control (control siPOOL) has no side effects on function or gene expression that we can detect as compared to untransfected cells. As compared to other commercially available single siRNAs, siPOOLs are much more effective, therefore, we can use less amounts of siPOOLs, which is very cost-efficient."

Dr. med. Peter Dietrich (Post-doctoral Scientist, Institute of Biochemistry Prof. Dr. Anja Katrin Bosserhoff Group FAU, Germany)

"The innovative solution of siPOOLs with 30 single siRNAs per pool is our first choice for conducting gene candidate approaches where gene loss-of-function has to be studied for dozens of genes. This approach is highly cost-effective and labor-saving when compared to gene silencing with single siRNAs or small pools of randomly selected siRNAs."

Dr. Peter-Christian Klöhn (Principal Investigator, MRC Prion Unit at UCL, Institute of Prion Diseases, London, UK)

FAQs about siPOOLs and siRNA Gene Silencing

A siPOOL uses high complexity pooling of ~30 siRNAs to dilute the off-target effects of each siRNA below experimental relevance. Proprietary siPOOL design algorithms ensure maximal transcript coverage and avoidance of paralogs, increasing gene knock-down efficiency and specificity respectively. Patented production of siPOOLs generate siRNAs of defined length and highest purity standards, minimizing risks of non-specific effects.

Other commercially available siRNA pools are either low complexity pools of 3-4 siRNAs, or stochastic pools of varying siRNA lengths. In contrast, siPOOLs are the first available high complexity pools of carefully selected and defined-length siRNAs.

siPOOLs are available at 5, 10 or 20 nmol per target gene. At a siPOOL concentration of 1 nM, 5 nmol is sufficient for at least 2250 wells of a 6-well plate or 45000 wells of a 96-well plate (refer siPOOL transfection protocol). Bulk orders of 10 or more siPOOLs are available at lower scales of 1-2 nmol. Please contact us to obtain custom quotations.

We need approximately 3-4 weeks to deliver a new siPOOL. For siPOOLs already in-stock, these are available within 2 weeks. Subject to sequence conditions, some siPOOLs may take longer to synthesize. siTOOLs Biotech will give notification if the delivery will be delayed.

Unique sequences ≥ 300 bases are frequently sufficient for the design of a siPOOL. A certain degree of sequence overlap between siRNAs is non-problematic with the condition that diversity of seed sequence is maintained. Please send your sequence information to info@sitools.de and we will inform you of the design feasibility and options.

All siPOOLs against coding genes are covered by a validation-inclusive guarantee. Here, ≥ 70% knock-down is guaranteed when the siPOOL is applied at a concentration of up to 10 nM and RNA quantified after 24 h. Optimal transfection conditions should be seen with a positive control siRNA in standard cell lines where the given target is expressed. If ≥ 70% knock-down is not seen, we will perform at no cost to the customer a re-design, re-synthesis, validation and shipping of a new siPOOL, subject to available sequence. As knock-down efficiency also varies with gene characteristics, improvement of knock-down efficiency with the new siPOOL is not guaranteed.

For siPOOLs against non-coding genes, a re-synthesis is performed if ≥ 60% knock-down is not achieved, subject to available sequence. Validation and shipping however are covered by the customer.

It is more challenging to silence long non-coding RNAs (lncRNA) by RNAi due to factors such as secondary structure, bound protein, or cellular localization limiting access to RNAi machinery. Depending on available transcript sequence, we will perform a round of siPOOL re-design and synthesis to target different regions of the lncRNA. However, validation of the siPOOL is best performed by the customer who is familiar with the properties of the lncRNA being studied.

Only 0.1 nmol positive (GAPDH/KIF11/INCENP) and negative control siPOOLs are free for testing. We welcome suggestions for frequently assessed genes and may include them as positive control genes in future.

Bulk orders of ≥ 30 siPOOLs are available at lower scales of 1 nmol. Pre-defined siPOOLs within the established siPOOL libraries are also available at smaller scales. Please contact us to obtain a quote.

RNAi machinery has been reported to be present in the nucleus and some siPOOLs against nuclear-localized RNAs (e.g. MALAT1, XIST) have worked with ≥ 70% knock-down efficiency. Cytosolic-localized RNAs however, are expected to be more efficiently targeted by RNAi.

Yes. siPOOLs are efficient at low nanomolar concentrations. This allows multiple siPOOLs to be combined in a single application to silence multiple genes with minimal risk of side-effects.

Up to four genes have been successfully silenced together with siPOOLs in this publication: Welsbie, D. S. et al. Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons. Neuron 94(6), 1142–1154.e6 (2017)

In standard cell lines (e.g. HeLa, Hek293, A549, MCF7), siPOOLs are typically applied at 1-3 nM with Lipofectamine transfection reagents. In difficult-to-transfect cells, higher concentrations may be required and alternative methods such as electroporation may be applied. We recommend performing a dose titration curve to determine the lowest concentration where knock-down efficiency plateaus. For long-term gene knock-down (> 3 days), a higher initial siPOOL concentration is recommended. A dose response optimization service is provided by siTOOLs Biotech where we perform a 7-point dose titration curve. Please contact us for more information.

Duration of siPOOL silencing is similar to other siRNAs and depends on the cell line and the target gene. Gene silencing can typically last from 4-7 days. Cells with a high proliferation rate or highly active genes may experience shorter durations of RNAi-mediated silencing. A re-transfection of siPOOLs or transfection at higher initial siPOOL concentrations may be performed to extend duration of silencing.

siPOOLs have successfully targeted selected isoforms with high specificity and efficiency. However, success depends on sequence availability unique to the isoform. Please send your transcript NCBI ID to info@sitools.de and we can check this for you. Cross-isoform/species selectivity siPOOL validation services are also provided.

We use a standard negative control siPOOL (30 siRNAs) that does not interact with human, mouse and rat genes. It has been tested in multiple cell lines and shows no significant impact on cell proliferation, apoptosis or cell morphology. Scrambled negative control siPOOLs can also be provided on request where target siRNA sequences are scrambled to avoid target interaction while maintaining %GC content.

Positive control siPOOLs are pre-validated siPOOLs that produce defined characteristics when introduced into cells, indicating successful transfection. Positive control siPOOLs available include siPOOLs against human KIF11 (3832) which produces mitotic arrest and observable cell death; human INCENP (3619) which produces enlarged cells observable under the microscope; and human GAPDH (2597) which produces no observable phenotype but exhibits strong decrease in GAPDH RNA levels verifiable by quantitative PCR. Positive control siPOOLs against mouse Gapdh (14433) and Kif11 (16551) are also available.

Yes, siPOOLs can be made to target any species. Please provide us with the target species, the host system and the target sequence.

No, you may apply the same optimized transfection conditions for siRNAs to siPOOLs.

There is a broad range of transfection reagents commercially available. For many common cell lines Lipofectamine works very well. However, transfection of cell types like primary macrophages or non-adherent cells present more challenges. siTOOLs Biotech offers a transfection optimization service in your provided cell line where we test 3 methods of transfection. Please contact us for more information.

siPOOLs are stable for at least 6 months when stored at -20°C though we have observed siPOOL activity even after several years. Splitting up larger volumes into multiple aliquots is strongly recommended to avoid multiple freeze thaw cycles.

Orders can be placed via our Webshop, directly at info@sitools.de or with our Distributors. Payments by bank direct transfer or credit card are preferred.

Yes, siPOOLs are not only used to routinely silence genes but as a validation approach for genes identified with other techniques such as CRISPR, single siRNAs or shRNAs. Please cite "siTOOLs Biotech" and our paper Hannus et al., 2014 when publishing with siPOOLs.

Yes. Further validation of siPOOL results can be performed with a rescue construct where a siPOOL-resistant version of the gene is expressed to restore function. More info.

We do NOT recommend deconvoluting a siPOOL as this would destroy the high specificity conferred by high complexity pooling. To further validate siPOOL results, we recommend using siPOOL-resistant rescue constructs.

siPOOLs are shipped in supension (10 mM Tris ph 8.0). siPOOLs have been tested to be stable for atleast 4 weeks at room temperature (RT), 37 °C for 1 week and at 50 °C for 24h., hence shipment delays in warm climates should not adversely affect siPOOL activity. 

siPOOLs are shipped world-wide. Customers from Japan, Korea, Singapore, Switzerland, Eastern Europe, Nordic Countries, USA and Israel can contact our distributors.

Yes. We have a siPOOL human kinase library containing 505 siPOOLs that can be used in high-throughput functional genomic screening to obtain reliable RNAi-derived hits, and siPOOL libraries for RNA binding proteins, E3 ligases, ubiquitinase, and GPCR.  For other custom library requests, please contact us.