FlexiTube siRNA

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.

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.

For the fixation of cells transfected with fluorescently labeled siRNA, we would suggest to perform the following protocol:

1. After transfection, remove the medium from the cells and wash the cells once with PBS.
2. Incubate the cells for 15 minutes at room temperature with 4% Paraformaldehyde (in PBS, pH 7.0). The cells should be completely covered by this solution (e.g., for a 96-well plate use 50 µl solution/well)
3. Wash the cells with PBS.

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)

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." 

 FlexiTube siRNA is available for human, mouse and rat genes at 5 nmol and 20 nmol scale. FlexiTube GeneSolution and FlexiTube siRNA at 1 nmol scale are available for human and mouse genes only

No, FlexiTube siRNA and FlexiTube GeneSolution products are only shipped lyophilized at room temperature. We do not offer these products in a different format.

Yes, you can select from HP Validated and HP GenomeWide siRNAs for the FlexiTube format. There is no price difference.

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.

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.

Yes, sequence information for FlexiTube is provided both for HP GenomeWide siRNAs and HP Validated siRNAs, in the enclosed paper documentation upon delivery.

No, the design of Flexitube siRNAs in GeneGlobe is not biased towards either the 3' or the 5' end of the target gene.

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.

http://www.sabiosciences.com/reversetransfection.php

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:

• 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. 

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.