TransIT-siQUEST® Transfection Reagent

High Knockdown and Low Toxicity Using TransIT-siQUEST® Reagent in CHO Cells Stably Expressing Firefly Luciferase. CHO-luc cells were grown in 24 wells plates and transfected with 25 nM of either a non-targeting siRNA or a anti-firefly luciferase siRNA using the indicated reagents with the volumes noted beneath each well. Luciferase expression, normalized to non-targeting siRNA control (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as percent cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph.

Inhibition of PPAR-alpha Expression in Primary Mouse Hepatocytes Using the TransIT-siQUEST® Reagent. Primary mouse hepatocytes were transfected with an anti-PPAR-alpha siRNA or a non-targeting control siRNA using the TransIT-siQUEST® Reagent. Twenty-four hours post-transfection, the amount of PPAR-alpha mRNA was measured relative to GAPDH mRNA levels using qRT-PCR and then scaled to the expression level of the specific target mRNA in the Opti-MEM® media only (untreated) control.

Delivery of Fluorescently-Labeled siRNA Using TransIT-siQUEST® Transfection Reagent. HeLa (70% confluence) cells in 12-well plates were transfected with TransIT-siQUEST® Transfection Reagent (3 μl/well) and Label IT® siRNA Tracker™ Cy-3-labeled siRNA duplexes (RED, 25 nM final concentration in the well). The cells were incubated 24 hours post-transfection then fixed and counterstained with counterstained with Alexa Fluor® 488 Phalloidin (GREEN) (Life Technologies). Confocal images were acquired on a Zeiss LSM 510 Confocal Microscope.

Efficient Target Gene Knockdown in Selected Cell Lines Using TransIT-siQUEST® Reagent. Reporter plasmids expressing both firefly and sea pansy luciferase were co-transfected into the indicated cell lines using TransIT® Plasmid Transfection Reagents. Targeted knockdown was achieved by transfection of an anti-firefly luciferase siRNA using the TransIT-siQUEST® Reagent. Twenty-four hours later, firefly luciferase expression was normalized to sea pansy luciferase expression and compared to the reagent alone control.