Two different FlexiTube products are available: FlexiTube siRNA and FlexiTube GeneSolution.
FlexiTube siRNA is a cost-effective solution for RNAi analysis of small numbers of genes. siRNAs are provided in 5 nmol or 20 nmol amounts for human, mouse, or rat genes, or in economical 1 nmol amounts for human and mouse genes.
FlexiTube GeneSolution is a gene-specific package of 4 preselected siRNAs (1 nmol) for a target gene. FlexiTube GeneSolutions enable researchers to use multiple siRNAs for each target ensuring reliable results. FlexiTube siRNA and FlexiTube GeneSolutions are designed using innovative HP OnGuard siRNA Design and are available at QIAGEN's GeneGlobe Web portal.
FlexiTube siRNA comes with a one-time-only replacement offer. If several FlexiTube siRNAs for the same target gene are ordered and at least 2 of them do not provide ≥70% target gene knockdown, QIAGEN will provide 2 additional siRNAs free of charge, once only. You will be asked to provide supporting data, demonstrating that the siRNA failed to knock down the target gene by at least 70% at the mRNA level under appropriate transfection conditions. Supporting data should include transfection efficiency data, quantitative silencing data, and data showing ≥70% knockdown of a positive control. This offer is valid for up to 6 months after the date of delivery.
Thousands of our human siRNAs have been experimentally verified by real-time RT-PCR analysis and shown to provide at least 70% knockdown. For these verified siRNAs, experimental details and information about the level of knockdown achieved are provided in GeneGlobe. These experimentally verified siRNAs are the result of the world's largest siRNA validation project, which was carried out by QIAGEN scientists. To find out more about this project, consult the publication Krueger, U. et al. (2007) Insights into Effective RNAi Gained from Large-Scale siRNA Validation Screening. Oligonucleotides 17, 237.
Advances in the siRNA design process ensure that QIAGEN's highly innovative and sophisticated HP OnGuard siRNA Design delivers potent and specific siRNA. siRNAs are designed using neural-network technology based on an extremely large set of data from RNAi experiments. siRNA design is then checked for homology to all other sequences of the genome using an up-to-date, nonredundant sequence database and a proprietary homology analysis tool. HP OnGuard siRNA Design incorporates many unique and advanced features (see table).
|Neural-network technology||siRNA design uses the BioPredsi neural-network, which is based on an extremely large RNAi data set.||1-3|
|The world's largest siRNA validation project||The design process was reinforced and improved by data from this project, in which QIAGEN scientists proved the effectiveness of thousands of siRNAs, were used to . A large number of druggable genome siRNAs have been proven to provide at least 70% knockdown during this project.||4|
|Homology analysis||Analysis uses a proprietary tool and an up-to-date, nonredundant sequence database.|
|Affymetrix GeneChip analysis||Genomewide analysis enabled development of siRNA design improvements that minimize off-target effects.|
|Up-to-date siRNA target sequences||Current data from NCBI databases ensure accurate design.|
|Asymmetry||siRNAs are designed with unequal stabilities of the base pairs at the 5' ends. This enables the antisense strand, which is less tightly bound at its 5' end, to enter RISC, while the sense strand is degraded. Asymmetry produces highly functional siRNAs and reduces the risk of off-target effects caused by the incorrect strand entering RISC.||5, 6|
|3' UTR/seed region analysis||Analysis uses intelligently weighted, multi-parameter searches for matches of the seed region of the siRNA antisense strand with the 3' untranslated region of unintended mRNA targets (see text for further explanation).||7-12|
|SNP avoidance||The RefSNP database is used to exclude siRNAs that span single nucleotide polymorphisms (SNPs). This increases siRNA potency, as an siRNA spanning a SNP will vary in its effectiveness.|
|Interferon motif avoidance||siRNAs are screened for multiple sequence motifs known to result in an interferon response. siRNAs with such motifs are rejected.||13, 14|
Several studies have shown that off-target effects may be caused by matches of the seed region of the siRNA antisense strand with the 3' untranslated region of unintended mRNA targets (see table). The seed region comprises 6 nucleotides in positions 2–7 of the antisense siRNA strand of the siRNA duplex. Matches such as these can contribute to downregulation of unintended targets due to the siRNA mimicking the action of an miRNA. siRNA designed at QIAGEN is analyzed for 3' UTR/seed region complementarity using a proprietary set of 3' UTR sequences derived from the human, rat, and mouse RefSeq databases. Each siRNA is aligned against these sequences to check for any homology that could contribute to miRNA-like, off-target effects.
Matches of 6 out of 6 nucleotides of the siRNA seed region with an unrelated target 3' UTR sequence are common and it is not necessary or practical to eliminate siRNAs showing such matches. More rarely, seed region matches in combination with 10 or more bases of additional homology are observed in an siRNA sequence. Such homologies have greater potential to result in off-target effects, and where possible these siRNAs are rejected in favor of others with less significant homology to unintended target genes.
For some targets, it is not possible to select siRNAs that do not show any such homologies. In these cases, EntrezGene IDs of the unrelated genes that could be unintended targets of the siRNA are provided at GeneGlobe. Observation of this type of homology does not necessarily mean that these genes will be affected by the siRNA. However, they can be considered potential unintended targets for follow up analysis, if warranted.
FlexiTube siRNA enables ordering of small numbers of siRNAs at cost-effective 1 nmol amounts (the minimum order is 4 siRNAs), or in 5 nmol and 20 nmol amounts. siRNAs are provided lyophilized in tubes.
For a streamlined RNAi solution for your target gene, simply enter details of the human or mouse genes of interest at the GeneGlobe Web portal. The search results will display the FlexiTube GeneSolution, which is the 4 recommended siRNAs for each gene provided in 1 nmol amounts. Published guidelines recommend redundancy experiments to ensure accurate reporting of results from RNAi experiments (Echeverri, C.J. et al. (2006) Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods. 3, 777; Echeverri, C.J. and Perrimon, N. (2006) High-throughput RNAi screening in cultured cells: a user’s guide. Nature Reviews Genetics 7, 373.). Redundancy experiments use several distinct siRNAs targeting different areas of the same mRNA to rule out off-target effects. An off-target phenotype is induced by the specific pattern of cross-silenced transcripts of an siRNA. Since this directly derives from the siRNA sequence, it is highly unlikely that several siRNAs with different sequences will share the same sequence-derived off-target effects. Confirming a phenotype with several distinct siRNAs is an easily applied and convincing way to show siRNA specificity. FlexiPlate GeneSolutions enable redundancy experiments for human and mouse genes. siRNAs are recommended by QIAGEN for each gene so that the optimal solution for your experiments is at your fingertips!
All siRNAs are provided with siRNA sequence information at no extra charge. Full sequence disclosure provides information for analysis of experimental results and verification of knockdown. siRNA sequences can be included in research publications, if required.
FlexiTube siRNAs in 20 nmol amounts are available with labels, including Alexa Fluor, fluorescein, rhodamine, Cy3, and Cy5 dyes, or modification options such as amino linkers, thio linkers, and phosphate modifications.
We recommend HiPerFect Transfection Reagent for low-throughput siRNA transfection and HiPerFect HTS Reagent for high-throughput siRNA transfection.
FlexiTube siRNA and FlexiTube GeneSolution are ideal for functional genomics or pathway analysis with small numbers of gene targets.
Please find a detailed description for the calculation of the silencing effect in QIAGEN News article 2006 e14 'Real-time RT-PCR for analysis of gene knockdown by RNAi - controls and calculations'.
Determine the transfection efficiency and identify the optimal siRNA concentration for the cell type. Assess the gene knockdown effect at mRNA level using real-time PCR. In some cases, you may need to assess mRNA levels at 48, 72, and 96 hours post-transfection. You may also want to include positive controls for both transfection and gene knockdown experiments.If the issue persists, send real-time PCR data and/or western blot data to QIAGEN Technical Service for further assistance
The siRNA sequences are the same. With FlexiTube siRNA 1 nmol scale, minimum 4 siRNAs required. It can be any human or mouse, any target, 4 different siRNAs, or the same siRNA. Whereas 1 nmol FlexiTube GeneSolution is a gene-specific package of 4 pre-selected siRNAs for the same target.
Cells transfected with Alexa-Fluor labeled siRNA still show detectable fluorescence 72 hours after transfection. Certain Alexa dyes, e.g. Alexa Fluor 546, are detectable up to one week after transfection. By comparison, when labeling siRNA with Rhodamine or Fluorescein, transfected cells should be monitored for transfection efficiency after 3-4 hours.
Since Alexa Fluor dyes are more photostable, more resistant to variable pH conditions while in transit through the cell, and much brighter than traditionally used fluorescent dyes, Alexa Fluor labeled HPP Grade siRNA is the ideal choice for monitoring transfection efficiency.
For data and additional details on using fluorescently labeled siRNA, refer to QIAGEN News article e20, 2004: 'Alexa Fluor labeled siRNA is highly effective for monitoring transfection efficiency'.
Different Flexitube siRNAs for the same target gene vary slightly in their nucleotide sequence and exact position along the target. All of them were selected by the unique HiPerformance siRNA Design Algorithm and thus have a very high likelihood for knocking down the target gene efficiently. Knockdown achieved with siRNA designed by the HiPerformance algorithm is usually greater than 70%.
Consequently, any of the FlexiTube siRNAs listed in GeneGlobe for a given gene can be chosen for knockdown experiments. We strongly recommend to try at least two siRNAs for independent confirmation that the observed effects are due to specific knockdown of the targeted gene.
Flexitube siRNA comes with a one-time–only replacement offer. If 2 or more HP GenomeWide siRNAs for the same target gene are ordered and none of the siRNAs result in gene silencing, QIAGEN will provide 2 additional siRNAs free of charge, once only. Please visit our website, or contact QIAGEN Technical Services for details and required proof data.
You just need to add sterile RNase-free water (which is supplied with your siRNAs) to resuspend your FlexiTube siRNA. You do not need to perform incubation steps at 96°C or at 37°C. Your siRNA comes preannealed and does not need any further preparation prior to use.
Some siRNAs have been deleted from our standard GeneGlobe offering. For those siRNAs, the entries in public databases may have changed and the transcript is no longer listed in those databases. In other cases, we have added siRNAs with a new design.
You can still find previously listed siRNAs in GeneGlobe by searching for the specific SI number (ordering number). See also FAQ 1366 on this topic.
QIAGEN siRNA is delivered as a stable, ready-to-use duplex and does not need to be deprotected, desalted, quantified, or annealed before use. Simply resuspend the lyophilized RNA in the sterile RNAse-free water provided and transfect. Instructions for preparing your siRNA are provided on the data sheet supplied with each siRNA shipment.
This decision is based on two key factors – the “transfectability” of the target cells, and the desired duration of the experiment.
siRNA molecules work well for high throughput, transient studies, with cells that are easily transfected. The limitations of using siRNA are two-fold. First of all, they do not work well with a few cell types that are extremely difficult to transfect. In addition, their use is restricted to experiments studying the impact of transient suppression of gene expression.
shRNA are carried within the context of a plasmid or viral-based vector, they can be engineered to carry a reporter gene. A reporter gene provides a straightforward readout, for carrying out transfection optimization studies. In addition, a fluorescent reporter gene (such as GFP) allows the use of fluorescence-activated cell sorting (FACS) to enrich for transfected cells. Alternatively, the vector may carry an antibiotic-resistance gene, which permits the selection of a stably transfected cell population. Another obvious advantage to using a vector-based shRNA construct is that it provides a renewable source of reagent for subsequent RNAi studies.
The key benefit of using viral-based shRNA delivery vectors is that they can efficiently deliver the shRNA into cells that are difficult (or impossible) to transfect. Additionally, viral transduction is a much more efficient process than transfection.
Yes, Northern Blot Analysis has been shown in the literature to detect siRNA-induced reduction of specific mRNA. Whether a Northern Blot will be sensitive enough to detect a mRNA under investigation mainly depends on the expression level of the respective gene in the untreated control.
You can find an example for this application in the reference "Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems". Caplen et al., PNAS 2001, vol. 98, no. 17, pages 9742-9747.
We recommend to perform real-time RT-PCR for exact quantification of mRNA expression levels.
For the fixation of cells transfected with fluorescently labeled siRNA, we would suggest to perform the following protocol:
Fixed cells can be stored at 4°C for a few days.
(Note: It is also possible to use chamber slides or object slides for this procedure. Object slides should be coated to provide better growing conditions for cells. Cells can be fixed as described above and then overlayed with embedding medium to allow investigation using a fluorescence microscope. Optimal conditions for this method need to be determined by the user)
|AllStars Hs Cell Death Control siRNA (20 nmol) 1027299
Negative Control siRNA (20 nmol) 1027310
Mm/Hs_MAPK1 control siRNA (20 nmol) 1027321
Negative controls are of critical importance, when performing RNAi studies, in order to confirm that any observed molecular and/or cellular changes are due to the sequence-specific RNAi event. Ideally you should use a scrambled artificial sequence that does not match any of the genes of the cell line/cell type being studied. It is important that appropriate experiments be carried out in advance to validate that the negative control siRNA under consideration has minimal impact on cell viability, proliferation, and global gene expression. The molar amount of negative control siRNA molecules used must be the same as the amount of experimental siRNA that are to be used in the knock down studies.AllStars Negative Control siRNA has been tested thoroughly for potential off target effects and has proven as suitable negative control siRNA already for many years.
Positive controls are also very useful, particularly when carrying out preliminary transfection optimization and/or assay development studies. As with the negative controls, positive controls should be experimentally validated in your model cell line of interest, at the appropriate siRNA concentration, prior to adopting them as acceptable controls. AllStars Cell Death Control siRNA is a phenotypic siRNA, which does not require tedious analysis steps.
Our R&D team has carried out an extensive study aimed at answering this question. A thorough evaluation of the major contributing factors essential to RNAi validation using qRT-PCR was carried out. A complete description of this study can be found at the following web address:
The conclusion of this study was that the three most important criteria to meet, in order to establish a reliable RNAi validation protocol, are as follows:
• Transfection efficiencies of 80% or higher
• Standard deviation in the technical replicate raw Ct values from the qPCR analyses should be no greater than 0.2.
• Carry out the experiment with no less than three biological replicates of each target gene-specific siRNA/shRNA and each negative control siRNA/shRNA.
"QIAGEN offers a variety of positive and negative control siRNAs. In addition, any of our functionally validated FlexiTube siRNAs are suitable positive controls for RNA interference (RNAi). Our AllStars Negative Control siRNAs, a randomly designed sequence with no known homology to mammalian genes, is the most thoroughly tested and validated negative control siRNA currently available. We strongly recommend to use our RNAi Human/Mouse Starter Kit, which includes HiPerFect Transfection Reagent, Allstars Negative Control siRNA, a positive control siRNA directed against human and mouse MAPK1 (HS/Mm_MAPK1 Control siRNA), and Allstars Hs Cell Death Control siRNA, a phenotypic control siRNA that allows monitoring gene silencing effects by light microscopy."
No, FlexiTube siRNA and FlexiTube GeneSolution products are only shipped lyophilized at room temperature. We do not offer these products in a different format.
However, to ensure accuracy, Custom siRNA Synthesis orders should be submitted in writing. Therefore you can use the HP Custom siRNA Order Form https://www.qiagen.com/products/genesilencing/customsirna/customsirnaorder.aspx?EmailOrdering=1.
Visit the RNAi Solutions page http://www.qiagen.com/products/rnai/default.aspx?r=2714 on our homepage for access to the Online Ordering Tool, and choose the order link for your product of interest.
Yes. Please see the QIAGEN News Article (2005 e3) "RNAi - a promising tool for target validation studies and therapeutics" to learn about the most recent advances in this field. It provides numerous references for the aspects of in vivo siRNA delivery and siRNA stability as well as in vivo RNAi in general. QIAGEN offers economical, high-purity siRNA compatible with in vivo animal delivery systems for reliable World-class RNAi solutions.
A number of siRNAs are located in the 3' untranslated region for optimized specificity of the siRNA sequences to their target gene. Since there are homologous sequences in the coding regions of other genes for many targets, it is usually easier to find highly unique siRNA sequences in the untranslated regions. Such design ensures highly specific silencing effects for the gene of interest.
HiPerFect Transfection Reagent is optimized for siRNA transfections and enables effective siRNA uptake and efficient release of siRNA inside cells, resulting in high gene knockdown even when using low siRNA concentrations. For high-throughput siRNA screenings HiPerFect HTS Reagent is a fast and effective newcomer specifically designed to focus on robustness and cost efficiency. For the transfection of shRNA.
Attractene Transfection Reagent should be used for the transfection of shRNA (short-hairpin RNA) vectors for gene silencing experiments to achieve high efficiency. Ease and flexibility of handling enables preparation and storage of transfection complexes making Attractene Reagent suitable for use with automated systems.
Fluorescently-labeled siRNA molecules have been shown to be transfected and processed in a manner that is indistinguishable from unlabeled siRNA. Therefore, these molecules serve as a powerful tool for simultaneously optimizing both siRNA transfection efficiency and the knock down of gene expression. FlexiTube siRNA and HP Custom siRNA is available at 20 nmol scale with different fluorescence labels including AlexaFluor dyes.
Quantitative gene expression analysis via a quantitative reverse transcription-PCR (qRT-PCR) assay is the gold standard for assessing the extent of gene expression knock down in an RNAi experiment. Alternative RNA detection methods, such as Northern blots, RNase protection assays, or end-point PCR, are not quantitative enough to reliably validate gene expression knock down. The RT2 qPCR Primer Assays are available for any gene in the human, mouse, or rat genome. Using these in combination with the pre-optimized RT2 SYBR Green Mastermixes, and the RT2 First Strand Kit provides the easiest, and most reliable, method for quickly evaluating the effectiveness of your gene expression knock down protocol.
Monitoring expression at the protein level via Western blot analysis, ELISA, immunofluorescence, or a functional assay is a critical step in confirming that a gene expression knock down experiment is ultimately resulting in decreased protein levels. However, it is very important to bear in mind that the kinetics of RNA knock down and protein knock down do not usually parallel one another. If the protein under study has a long half-life, then changes in protein level will take much longer to occur than changes in the RNA level. Additionally, it is important to keep in mind that the quality of any antibody-based protein detection assay is dependent upon the quality of the antibody being used.
Phenotypic change in the cells following siRNA delivery, can sometimes be a useful readout for monitoring the effectiveness of an RNAi experiment. AllStars Cell Death Control siRNA is a phenotypic siRNA that has been developed to work in virtually all cell times, as it consists of a blend of siRNAs addressing different vital pathways.
Here are examples of references that describe the inhibition of gene expression by siRNA in Xenopus and Zebrafish:
You will receive the sequences of the FlexiTube siRNAs on the data sheet along with your order.
A graphic representation of the position of HP GenomeWide siRNAs along the target sequence is available for numerous genes on our website. It can be accessed via GeneGlobe after pulling up the target gene of interest, clicking on the 'Gene Symbol' link for the species of interest, and then clicking the link under 'Product name' for details on the gene product of interest.
The Alexa Fluor 488 fluorophore is brighter and more photostable than other fluorescent labels. It is tolerant of pH changes within a wide range, making it very stable in living cells. For example, fuorescence microscopy of cells transfected with Alexa Fluor- and FITC-labeled siRNAs after 24 hours showed that the signal of the Alexa Fluor fluorophore was much more persistent than that of FITC.
The most accurate method for validating RNA interference is to carry out qRT-PCR on RNA isolated from an enriched or selected population of transfected cells. When carrying out these assays, special care should be taken to insure that highly reproducible biological replicates, as well as technical replicates of the qRT-PCR analysis are performed. This will enable the reliable detection of the roughly 1.75 to 2.0 threshold cycle differences between gene-specific and negative control siRNA/ shRNA transfected cells, which are typically seen in RNAi experiments.
In the case of difficult cell types or weak silencing effects, it may be helpful to increase the final siRNA concentration during transfection. This can be achieved simply by using larger amounts of FlexiTube siRNA Premix for transfection.