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NOTE: Each kit contains sufficient quantities to label 50 µg siRNA.
Each Kit contains Label IT® siRNA Tracker Reagent, Label IT® Reconstitution Solution, 10X Labeling Buffer A, and siRNA Dilution Buffer.
For more information on siRNA delivery in vivo , see the following references:
Lewis et al.(2002) Nature Genetics 32: 107-107.
Zhang et al.(1999) Human Gene Therapy 10: 1735-1737.
Liu et al.(1999) Gene Therapy 6: 1258-1266.
Hagstrom et al.(2004) Molecular Therapy 10: 386-398.
Label IT® siRNA Tracker Kits. The Label IT® siRNA Tracker labeling reagents are composed of three regions: the label (a fluorophore or biotin) (green), the linker (yellow) which facilitates electrostatic interactions with the siRNA and miRNA and the reactive alkylating group (blue) that covalently attaches the Label IT® reagents to any reactive heteroatom within the siRNA or miRNA. Attachment of the Label IT® Reagent to siRNA or miRNA is optimized and does not alter their structure or affect downstream target knockdown performance. The labeled siRNAs or miRNAs can be both visualized after transfection by fluorescence microscopy and simultaneously employed to knockdown target gene expression.
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.
Delivery of Labeled siRNA to Mouse Liver. (A) siRNA (25 µg) was labeled with Label IT® siRNA Tracker Cy®3 Kit and delivered to mice through the tail vein using either low volume injection conditions (100 µl) hydrodynamic injection conditions (2 ml over 6-8 seconds). (B) Mouse livers were harvested 30 minutes after injection, fixed in paraformaldehyde, sectioned (10 µm thick) and analyzed. A representative hepatocyte (h) and sinusoid (s) are indicated in each panel. The hydrodynamic injection of siRNA results in siRNA uptake by the majority of the hepatocytes in the liver.
Label IT® Excitation and Emission Spectra
Technical Report: High Efficiency siRNA Delivery In Vitro
Technical Report: Delivery Tools for Neurobiology
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