FlexiTube siRNA Premix is an optimized mixture of siRNA and transfection reagent. Premixing of siRNA and reagent saves time by eliminating mixing and complex formation protocol steps. In addition, use of FlexiTube siRNA Premix removes the effort involved in estimating optimal siRNA-reagent ratios. FlexiTube siRNA Premix is available for human and mouse genes.
The ratio of reagent to siRNA in FlexiTube siRNA Premix provides highly efficient transfection and gene knockdown (see figures " Rapid, efficient knockdown" and " Phenotype analysis after knockdown").
FlexiTube siRNA Premix contains FlexiTube siRNA, which is designed using highly innovative and sophisticated HP OnGuard siRNA Design to deliver high potency and specificity. siRNAs are designed using neural-network technology based on a large data set 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 that incorporates exciting design features.
FlexiTube siRNA Premixes come with a one-time-only replacement offer. If several FlexiTube siRNA Premixes 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.
FlexiTube siRNA Premix is ready-to-use for cell transfection. Simply add to cells and incubate. This is a significant time and labor saving compared with a typical transfection experiment in which siRNA is diluted, mixed with transfection reagent, and then the mixture is incubated to allow siRNA-reagent complex formation prior to adding the complexes to the cells and incubating.
With FlexiTube siRNA Premix, RNAi experiments get off to a faster start, as there is no need to optimize siRNA to reagent ratio. Tedious optimization experiments involving multiple transfections are minimized or eliminated, as FlexiTube siRNA Premix provides siRNA and reagent premixed at an optimal ratio.
Multiple transfections can be performed from a single FlexiTube siRNA Premix. This reduces variability, enabling consistency across experiments and more reliable results.
FlexiTube siRNA Premix is ideal for functional genomics or pathway analysis with small numbers of gene targets.
|HP OnGuard design
|Target sequence provided
|siRNA per target gene
|Number of transfections
|50 standard transfections in 24-well format
|for validated siRNAs
|Scale or yield
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
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."
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.
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:
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.