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Confirming RNAi Effects Is Now Easier Than Ever

To validate any RNAi experiment, target mRNA reduction should be confirmed and quantitated. Validation, using a positive control siRNA that is known to be effective, is required for optimization of transfection parameters. Validation is also necessary for assessment of the effectiveness and potency of any siRNA sequence. Finally, target mRNA levels after siRNA delivery should be quantitated so that phenotypes can be correlated with the extent of mRNA knockdown.

Several different methods can be used to monitor target mRNA levels following siRNA transfection, including Northern analysis, nuclease protection assay, gel-based RT-PCR, and real-time PCR. Of these methods, real-time PCR has distinct advantages: it is the most sensitive of all of the RNA detection methods, and it can be completed in the least amount of time if optimized primer and probe sets are used.

For real-time PCR analysis of target mRNA levels for RNAi experiments, Ambion scientists recommend TaqMan Gene Expression Assays (formerly called Assays-On-Demand) from Applied Biosystems. The TaqMan Gene Expression Assays include an extensive collection of premade TaqMan primer and probe sets for quantitative, real-time PCR gene expression studies. They are considered the gold standard for gene expression analysis. Pre-designed TaqMan Gene Expression Assays are available for the human, mouse, and rat genomes, complementing Ambion's Silencer Pre-designed, Validated an d Library siRNA products. Each gene specific assay is designed to work under universal thermal cycling conditions and does not require optimization prior to use.

Figure 1. Knockdown of Survivin with a Silencer Validated siRNA and Verification with TaqMan Gene Expression Assays. HeLa cells were transfected with 30 nM of a Silencer Validated siRNA targeting survivin (BIRC5) or Silencer Negative Control #1 in quadruplicate. Forty-eight hours later, two sets of samples were stained with DAPI and analyzed by fluorescence microscopy. Total RNA was isolated from the other two sample sets using RNAqueous MAG-96 and then converted to cDNA. Target cDNA levels were analyzed in duplicate using a TaqMan Gene Expression Assay for survivin (Hs00153353_m1). Data shown here represent duplicate assays from single transfections. Survivin mRNA levels were reduced 80% in the survivin siRNA treated cells relative to control siRNA treated cells. Knockdown of survivin induced morphological changes to cell nuclei. The insets show representative cells from the survivin and control siRNA treated samples.

To make it easier than ever to find TaqMan Gene Expression Assays for your siRNA experiment, Ambion's online siRNA Database now provides links to individual gene expression assays on the Applied Biosystems' website. The siRNA Database provides information about Ambion's Silencer Pre-designed siRNAs targeting >98% of all human, mouse, and rat genes in NCBI's RefSeq database, as well as Silencer Validated siRNAs, which have been functionally proven to reduce target mRNA levels by 70%. To find siRNAs and TaqMan Gene Expression Assays to your gene of interest, search our siRNA Database. You can search by gene name, RefSeq accession number, or other accession number, and will be presented with comprehensive solutions for both gene silencing and the downstream analysis of the silencing effect.

Applied Biosystems is a registered trademark of Applera Corporation. The PCR process and 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. TaqMan is a registered trademark of Roche Molecular Systems, Inc.

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Ordering Information
Cat# Product Name Size 16700 Silencer Pre-designed siRNA, standard purity 20 nmol 16702 Silencer Pre-designed siRNA, standard purity 40 nmol 16704 Silencer Pre-designed siRNA, standard purity, annealed 20 nmol 16706 Silencer Pre-designed siRNA, standard purity, annealed 40 nmol 16800 Silencer Pre-designed siRNA, HPLC purified 40 nmol 16802 Silencer Pre-designed siRNA, HPLC purified 160 nmol 16804 Silencer Pre-designed siRNA, HPLC purified, annealed 40 nmol 16806 Silencer Pre-designed siRNA, HPLC purified, annealed 160 nmol 16808 Silencer Pre-designed siRNA, HPLC purified 20 nmol 16810 Silencer Pre-designed siRNA, HPLC purified, annealed 20 nmol 16900 Silencer Pre-designed siRNA, PAGE purified 25 nmol 16904 Silencer Pre-designed siRNA, PAGE purified, annealed 25 nmol


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