Cat. No. / ID: 1027411
FlexiPlate siRNA provides highly flexible RNAi screening and is available at 0.1 nmol, 0.25 nmol, and 1 nmol scales in 96-well plates, and at 0.1 nmol and 0.25 nmol scales in 384-well plates for a choice of target genes. For maximum flexibility, siRNAs can be selected and plate layout specified at the GeneGlobe Web portal. Lists of preselected siRNAs are also available for many gene families. siRNAs have been designed using HP OnGuard siRNA Design, which incorporates neural network technology, proprietary homology analysis, and advanced features, such as 3' UTR/seed region analysis, asymmetry, SNP avoidance, and interferon motif avoidance.
Note: QIAGEN does not provide FlexiPlate siRNA in pools.
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. 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. >
FlexiPlate siRNA allows design of RNAi screening experiments to suit specific requirements. siRNAs for any human or mouse gene can be selected as well as any positive and negative controls required. With the user-friendly Web interface, siRNAs and controls can be placed in any of the wells of a 96-well or 384-well plate, or plate layout can be selected from a range of predefined layout patterns. siRNAs are provided in 0.1 nmol, 0.25 nmol, or 1 nmol scales, enabling economical screening using low or higher siRNA amounts, as required. The 0.1 nmol, 0.25 nmol, and 1 nmol scales are available in 96-well plates. The 0.1 nmol and 0.25 nmol scales are available in 384-well plates.
The GeneGlobe Web portal makes it easy to search for siRNAs for genes of interest and to arrange plate layouts to suit screening experiments. Lists of gene names, Entrez Gene IDs, RefSeq IDs, siRNA names, or catalog numbers can be uploaded, making the process of plate ordering fast and easy. Order plates immediately or save for later changes and ordering. Easily download plate layouts for your records.
FlexiPlate siRNA enables RNAi applications including:
|Design||Predesigned/HiPerformance siRNA Design Algorithm|
|Target sequence provided||Yes|
|Scale or yield||0.1 nmol, 0.25 nmol, 1 nmol|
We cannot provide FlexiPlate siRNAs at a higher final concentration. To obtain more concentrated siRNA solutions, individual lyophilized siRNAs can be resuspended in smaller amounts of sterile RNase-Free water. See FAQ 1360 for additional details on recommended resuspension volumes for various scales of FlexiPlate siRNA.
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
We do not expect a difference in performance of the siRNAs in case FlexiPlate siRNA was accidentally redissolved in siRNA Suspension Buffer. However, we have not tested this and do not have any proof data.
With FlexiPlate siRNA, you receive a CD with an Excel file that contains the following information (example):
We have updated GeneGlobe and have exchanged several siRNAs with new designs. To find and order siRNAs from your set in GeneGlobe, search by using the siRNA catalog number (e.g., SI00000203). If you search by entering the gene name or accession number, only the new siRNA designs will be shown.
Individual siRNAs from the QIAGEN HP siRNA Sets can be reordered in FlexiPlate format by entering the corresponding GeneGlobe catalog number (SI number) for the specific siRNA. SI number information for each siRNA is provided on the annotation CD which is included in the shipment of the siRNA Set.
You can add the following controls to your FlexiPlate siRNA plate: AllStars Negative Control siRNA, AllStars Cell Death Control siRNA, Negative Control siRNA, Human GAPDH siRNA, Human Beta-Actin siRNA, Human and mouse MAPK1 siRNA, Human or mouse Lamin A/C siRNA, Mouse AKT1 siRNA, or other siRNAs from GeneGlobe, such as HP Validated siRNAs.
FlexiPlate siRNAs will be supplied in NUNC Polystyrene V bottom plates. NUNC Polystyrene plates can be ordered from Thermo Scientific Company.
Other plates like ABgene AbGene Polypropylene plates can be used but ONLY upon request
From the home page go to>Products>Genes & Pathways>Plate Designer. Alternatively, Click on this link Upload an Excel File .
Ordering FlexiPlate siRNA
Use this form to order FlexiPlate siRNA.
Order Form for FlexiPlate siRNA
Along with the Word Order Form, you must also send plate layout information. If you have configured your plates on our Website, download the plate data and send it with the Order Form. Alternatively, you can use the Excel template below to create your own spreadsheet. For help filling in the template, see our detailed siRNA plate template instructions.
You can also use this template to create your own file for uploading to our Website, where you can configure and order your plates online.
FlexiPlate siRNA buffer is 1x annealing buffer:
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.
FlexiPlate siRNA is available in 3 different amounts: 0.1 nmol, 0.25 nmol, or 1 nmol per siRNA. Up to 4 human or mouse siRNAs can be selected per gene.
FlexiPlate siRNA is provided lyophilized in 96-well plates and is shipped at room temperature. siRNAs can be arranged in plates using the drag-and-drop tool at the GeneGlobe Web portal. Special formats are available on request: delivery in 384-well plates, delivery in solution and aliquoting.
We strongly recommend our standard method of shipment for FlexiPlate siRNAs in lyophilized form at room temperature. Delivery in solution on dry ice is possible as an exception only and has to be requested specifically when placing the order.
0.1 nmol scale – add 10 ul of 10uM stock solution.
120 transfections in 96 well plates at 5 nM final concentration OR
60 transfections at 10nM final concentration.
0.25 nmol scale - add 25 ul of 10uM stock solution.
300 transfections in 96 well plates at 5 nM final concentration OR
150 transfections at 10nM final concentration.
1.0 nmol scale - add 50 ul of 20uM stock solution.
1200 transfections in 96 well plates at 5 nM final concentration OR
600 transfections at 10nM final concentration.
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."
Labeled siRNAs must be ordered separately. They cannot be provided on the same plate with FlexiPlate siRNA.
The minimum order number is 36 in 96 well plates or on average 36 siRNAs per plate. The minimum average number of siRNAs per plate is 36. Thus, with 40 siRNAs, you would receive the siRNAs on one plate. For 72 siRNAs, you can split the siRNAs on 2 plates with 36 siRNA on each plate. If you order 150 siRNAs, you can for example get 3 plates with 50 siRNAs each.
To summarize: The minimum number of siRNAs for each catalog Number (or siRNA Sacle -1 nmol, 0.25 nmol or 0.1 nmol) is 36. Within a specific scale, plate can have less than 36 siRNAs, if the minimum average number of siRNAs per plate is 36 siRNAs.
You can search for FlexiPlate siRNAs and design your plate layout online and then download it to an Excel file. Send the Excel file or the ID number of the Excel file to QIAGEN by fax or e-mail. Please also send the Word Flexiplate siRNA Order Form which includes shipping and billing information. Find out more about how to order if you cannot order online.
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.
FlexiPlate siRNAs are preannealed and dried down from a 10 µM solution in buffer. They should be resuspended in sterile RNase-free water only (you do not need siRNA buffer to resuspend the siRNAs).
To achieve a final siRNA stock concentration of 10 µM, resuspend:
Mix gently and cover the plate with new sealing film. Allow siRNAs to dissolve for 30 minutes at 4°C with occasional shaking or gentle vortexing.
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.
Only one scale of siRNAs is available per FlexiPlate. If different scales are required, siRNAs have to be ordered on separate FlexiPlates. When ordering different scales on different plates, note that the average number of siRNAs per plate has to be a minimum of 36.
Please see FAQ 1370 for an example and further details.
The standard turnaround time for FlexiPlate siRNA orders is 8 to 10 days. Turnaround times for special orders requiring pooling or aliquotting will differ, and will be communicated to the enduser upon ordering.
To locate and order specific FlexiPlate siRNAs you can upload a list of search terms such as:
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.
Flexiplate siRNAs are delivered annealed and lyophilized. Each plate is accompanied by an instruction sheet detailing how to reconstitute the siRNAs in the appropriate volume of RNase-free water, as well as a CD containing the siRNAs IDs, sequences information and gene annotations. See FAQ 1363.
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.