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How do siPOOLs increase specificity and efficiency of gene knock-down?
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.
What is the difference between your product and the other commercially available pools of siRNAs?
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.
How much do I get if I order a siPOOL and how many reactions can I run with it?
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.
How long does it take from placing an order to delivery?
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.
What is the minimum sequence length that can be targeted by a siPOOL?
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 email@example.com and we will inform you of the design feasibility and options.
What is guaranteed with a siPOOL purchase?
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.
Why is validation not included for siPOOLs against long non-coding RNA?
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.
Can you send a free sample for testing?
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.
Can I order smaller scales at a lower price for testing or screening?
Bulk orders of ≥ 10 siPOOLs are available at lower scales of 1-2 nmol. Pre-defined siPOOLs within the human kinase siPOOL library and the siPOOL cancer toolbox are also available at smaller scales. Please contact us to obtain a quote.
Do siPOOLs work for nuclear-localized RNA?
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.
Can siPOOLs be combined to knock down several genes at the same time?
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)
What concentration of siPOOLs should I use?
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.
How long does siPOOL-mediated silencing last?
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.
Can siPOOLs target specific isoforms?
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 firstname.lastname@example.org and we can check this for you. Cross-isoform/species selectivity siPOOL validation services are also provided.
What is your negative control siPOOL?
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.
What is your positive control siPOOL and what is the read-out?
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.
Can siPOOLs target other species aside from human, mouse and rat?
Yes, siPOOLs can be made to target any species. Please provide us with the target species, the host system and the target sequence.
Are transfection conditions different from siPOOLs and other siRNA reagents?
No, you may apply the same optimized transfection conditions for siRNAs to siPOOLs.
Which transfection reagent do you recommend?
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.
What is the shelf-life of a siPOOL?
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.
Can siPOOLs be used as a published validation approach?
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., Nucleic Acids Res, 2014 when publishing with siPOOLs.
Is there a way to further validate siPOOLs e.g. performing a rescue?
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.
Should I deconvolute a siPOOL to test siRNAs individually?
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.
How are siPOOLs shipped? Are they stable at room temperature?
siPOOLs are shipped freeze-dried in powder form. This makes them stable at room temperature until resuspension in nuclease-free water, which is provided. siPOOLs have been tested to be stable for at least two weeks at room temperature and at least a day at 50°C, hence shipment delays in warm climates should not adversely affect siPOOL activity.
Are siPOOL libraries available for functional genomic screening?
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. siPOOL libraries for RNA binding proteins and E3 ligases and deubiquitinases will become available soon. For other custom library requests, please contact us.
What is the difference between raPOOLs and other commercial biotinylated probes?
Other commercial suppliers typically offer biotinylated probes individually. raPOOLs in contrast, are sold as a highly complex mixture of 30 single-stranded biotinylated DNA probes. The high complexity of raPOOLs increases chances of association with the target RNA, making RNA pulldown more robust and reliable.
How are raPOOLs designed?
The 30 probes within a raPOOL are tiled across the target RNA of interest. siTOOL’s design algorithms additionally incorporate off-target filters to minimize non-specific binding, and optimized thermodynamics to increase probe binding affinity. Custom specifications such as targeting selected isoforms or special species can also be incorporated on request.
What are the characteristics of the biotin attachment to raPOOL probes?
The biotin is at the 3’ end of the single-stranded DNA probes that make up the raPOOL. The biotin is linked with an 11 atom spacer arm to avoid stearic hindrance.
How long does a raPOOL delivery take?
raPOOLs typically take 2-3 weeks to be delivered. siTOOLs Biotech will give notification if the delivery will be delayed.
How do I perform an RNA affinity purification with raPOOLs?
A protocol on RNA affinity purification with raPOOLs can be found under Resources > Protocols
What proteins/nucleic acids are known to interact with MALAT1, the positive control raPOOL?
The MALAT1 raPOOL was noted by customers to successfully pull down published proteins that include SR splicing factors (SRSF 2, SRSF3, SRSF5) and heterogeneous nuclear ribonucleoproteins (HNRNPC, HNRNPPF). Publication of MALAT1 interaction with SRSFs:
Tripathi, V et al. (2010) The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation. Mol. Cell. 39, 925–938
The MALAT1 raPOOL was also noted by customers to enrich published DNA targets HEXIM1, RNF40, PNN and PS2 in PC-3 cells. Publication of MALAT1 interaction with chromatin targets:
West, J. A et al. (2014) The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol. Cell. 55, 791–802
Is it necessary to perform sonication prior to the RNA pulldown?
Sonication is performed to thoroughly disrupt cells and randomly shear nucleic acids to increase efficiency of their pulldown, hence improving detection sensitivity. It is recommended and sonication conditions have to be optimized depending on the number and cell type used. Useful blogpost: Tips for optimizing RNA affinity purification
Is it necessary to perform cross-linking prior to the RNA pulldown?
Cross-linking is performed to stabilize molecular interactions before the pulldown. It may not always be necessary and depends largely on the RNA of interest as well as the goal of the pulldown (i.e. characterization of DNA vs. protein interactors). Different forms of cross-linking such as formaldehyde, glutaraldehyde and UV can be used. Useful blogpost: Tips for optimizing RNA affinity purification
What are the key factors determining RNA pulldown efficiency?
In terms of importance, starting material is key. Sufficient amounts of RNA need to be enriched to detect proteins. Hence, target RNAs that are lowly abundant may require the analysis of large numbers of cells which can pose challenges in terms of cell culture storage and handling capacity. Other notable factors that influence enrichment efficiency include cross-linking and sonication conditions, salt concentrations in the buffer, hybridization time (overnight hybridization has been reported to vastly improve RNA enrichment), and probe concentrations. Useful blogpost: Tips for optimizing RNA affinity purification
How are raPOOLs shipped? Are they stable at room temperature?
raPOOLs are shipped freeze-dried in powder form. This makes them stable at room temperature until resuspension in nuclease-free water, which is provided. raPOOLs have been tested to be stable for at least two weeks at room temperature and at least a day at 50°C, hence shipment delays in warm climates should not adversely affect raPOOL activity.
How many pulldown experiments can I perform with 10nmol raPOOL?
This is dependent on the target RNA of interest. With the recommended starting amount of 100pmol of raPOOL per ml of lysate, 10 nmol should be sufficient for at least 90 pulldown experiments.
Can raPOOLs work for mRNA?
Yes, the same principle of RNA enrichment with raPOOLs applies as well to messenger RNA.
What is the shelf-life of a raPOOL?
raPOOLs are stable for at least 6 months when stored at -20°C in nuclease-free water. Splitting up larger volumes into multiple aliquots is strongly recommended to avoid multiple freeze thaw cycles.
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