FlexiPlate siRNA

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.

You can upload an Excel file with your gene list for FlexiPlate siRNA, including gene-specific information, to GeneGlobe. At the GeneGlobe site, an example file with details on how to enter information is provided.

FlexiPlate siRNAs will be supplied in NUNC Polystyrene V bottom plates.  NUNC Polystyrene plates can be ordered from Thermo Scientific Company.

• Nunc MicroWell* 96-Well Microplates catalog # 249940.
• Nunc* 384-Well Polystyrene Plates catalog # 265203.

http://www.thermoscientific.com/ecomm/servlet/home?storeId=11152&langId=-1

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.

     Excel Template (CSV version)

FlexiPlate siRNA buffer is 1x annealing buffer:

• 30 mM HEPES (pH 7.4)
• 100 mM Potassium Acetate
• 2 mM Magnesium Acetate

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.

We do not provide FlexiPlate siRNAs in customer-specific plates for single plate orders. For large orders, there is the possibility to switch to other plates. Please inquire for details by contacting QIAGEN Technical Service.

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.

Yes, you can order HP GenomeWide siRNAs and HP Validated siRNAs with the Flexiplate siRNA format. There is no price difference between HP GenomeWide siRNAs and HP Validated siRNAs ordered as FlexiPlate siRNA. For HP Validated siRNAs, no sequence information is provided.

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:

http://www.sabiosciences.com/manuals/shRNAwhitepaper.pdf

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.

FlexiTube siRNA, FlexiTube GeneSolution, FlexiTube siRNA Premix, FlexiPlate siRNA, and GeneFamily Lists siRNAs can be ordered by catalog number over the telephone.

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:

• 0.1 nmol siRNA in 10 µl
• 0.25 nmol siRNA in 25 µl
• 1 nmol siRNA in 100 µl

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:

• Entrez Gene IDs (e.g., 100)
• Gene symbols (e.g., ADA)
• RefSeq IDs (e.g., NM_000022)
• Catalog numbers (e.g., SI00000203)
• siRNA names (e.g., Hs_ADA_1)

Here are examples of references that describe the inhibition of gene expression by siRNA in Xenopus and Zebrafish:

• Wargelius A, Ellingsen S, Fjose A. Double-stranded RNA induces specific developmental defects in zebrafish embryos.  Biochem Biophys Res Commun. 1999 Sep 16; 263(1): 156-61
• Zhou Y, Ching YP, Kok KH, Kung HF, Jin DY. Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.  Nucleic Acids Res. 2002 Apr 1; 30(7): 1664-9
• Zhao, Y Cao, M Li, and A Meng Double-stranded RNA injection produces nonspecific defects in zebrafish. Dev Biol, Jan 2001; 229(1): 215-23."

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.

QIAGEN has performed intensive validation experiments for FlexiTube and FlexiPlate siRNAs resulting in more than 3700 experimentally tested siRNA now available at GeneGlobe.

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.