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siRNA Mediated Gene Silencing

siRNA

Apart from their natural occurrence, exogenous sequences of siRNAs can be designed and introduced into cells through transfection to knock down relevant gene expression. This application serves as a tool to elucidate genetic pathways, determine protein function, or uncover new gene targets for biotherapeutic and pharmaceutical applications. Larger libraries of these siRNA molecules are also available to perform larger genome RNAi analysis via high-throughput RNAi screening.

EXOGENOUS SEQUENCES OF siRNAs...

can be designed and introduced into cells through transfection to knock down relevant gene expression.

Because siRNA differs in size and structure from plasmid DNA, transfection reagents can be optimized and formulated separately for delivery of these RNA molecules. In addition, delivery of siRNA to the cytoplasm for incorporation into the RISC complex is sufficient for gene knockdown. Selection of the appropriate transfection methodology or reagent must first be considered, followed by further optimization for efficient siRNA delivery and subsequent gene knockdown. Optimization of several experimental parameters is key to achieving the highest efficiency siRNA transfection and corresponding effective knockdown of target gene expression. For detailed information on the optimization of siRNA transfections, please see Tips from the Bench - Optimize siRNA Transfection.

A high and reliable level of knockdown can be achieved through siRNA transfection via a variety of quality transfection reagents and validated siRNA libraries. However, a few disadvantages with siRNA mediated knockdown are the chances of off-target effects and transient knockdown through siRNA dilution after multiple cell divisions. A viable alternative in this scenario is shRNA mediated gene-silencing.

If direct tracking of siRNA delivery is desired, siRNAs can also be labeled with different fluorophores using Label IT® siRNA Tracker Intracellular Localization Kits. See figure below.

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Successful Delivery and Functional Knockdown Using Labeled siRNA.

Successful Delivery and Functional Knockdown Using Labeled siRNA. (A) HeLa cells in 12-well plates were transfected at 70% confluence with TransIT-TKO® Transfection Reagent (3 μl/well) and Label IT® siRNA Tracker Fluorescein-labeled siRNA duplexes (GREEN, 50 nM final concentration in the well). The cells were incubated 24 hours post-transfection then fixed and counterstained with TO-PRO®-3 (nuclei, BLUE) and Alexa Fluor® 546 Phalloidin (actin, RED). Confocal images were acquired on a Zeiss LSM 510 Confocal Microscope. (B) TransIT-TKO® Transfection Reagent was used to transfect anti-firefly luciferase siRNA into CHO-Luc cells stably expressing firefly luciferase. The siRNA was either unlabeled or labeled with Label IT® siRNA Tracker Cy®3, Cy®5, Fluorescein, or CX-Rhodamine Reagents. Bars indicate the percent firefly luciferase expression 24 hours after delivery of 5 nM anti-firefly luciferase siRNA.

TransIT-X2® Dynamic Delivery System is the reagent of choice for delivering siRNA. TransIT-X2® delivers both plasmid DNA and/or siRNA efficiently and can be used for co-transfecting plasmid DNA and siRNA. TransIT-TKO® and TransIT-siQUEST® Transfection Reagents can also effectively transfect siRNAs into mammalian cells in culture. Ideally, all three reagents should be tested in parallel to determine the best solution for your application. Depending on the cell type, one reagent may have superior performance over the others. For cell-type specific recommendations, please consult the Reagent Agent® transfection database.

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For more information on transfection for RNAi applications, additional resources can be found on the Mirus Bio website:

Optimize siRNA Transfection
Deliver microRNA (miRNA) Effectively
Reverse Transfection Protocol for siRNA/miRNA