TransIT-TKO® Transfection Reagent

Fig 1. High Efficiency Endogenous Knockdown in iCell® Cardiomyocytes. The TransIT-TKO® Transfection Reagent was used to transfect iCell Cardiomyocytes (Cellular Dynamics International) plated at a density of 136,500 cells per well of a 12-well plate pre-coated with fibronectin. Seven days post-plating triplicate wells were transfected with TransIT-TKO (3-5 ml per well) and non-targeting control siRNA or GAPDH targeting siRNA (50nM per well). Seventy-two hours post-transfection, the amount of GAPDH mRNA was measured relative to 18s rRNA mRNA levels using qRT-PCR and then scaled to the expression level of the non-targeting control siRNA.  Error bars represent the standard error of the mean (SEM) of three independent complexes. See more information on stem cell applications.

Figure 2. Delivery of Fluorescently-Labeled siRNA using TransIT-TKO® Transfection Reagent. HeLa (70% confluence) cells in 12-well plates were transfected 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) (Life Technologies) and Alexa Fluor® 546 Phalloidin (actin, RED) (Life Technologies). Confocal images were acquired on a Zeiss LSM 510 Confocal Microscope.

Figure 3. High Efficiency Knockdown and Low Toxicity Using TransIT-TKO® Reagent in HeLa cells. Firefly and sea pansy luciferase reporter vectors were co-transfected into HeLa cells using TransIT®-LT1 Reagent. Cells were incubated for at least 4 hours, split into 24-well plates, allowed to adhere and transfected with 25 nM of either a non-targeting siRNA or an 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 % 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.

Figure 4. Efficient Knockdown of Endogenous Genes in Primary Hepatocytes Using TransIT-TKO® Reagent. Primary mouse hepatocytes were transfected with the indicated siRNAs or a non-targeting control siRNA using the TransIT-TKO® Reagent. Twenty-four hours post-transfection, the amount of each 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 cells alone (untreated) controls.

Figure 5. Knockdown of Endogenous Genes Using TransIT-TKO® Reagent. Various cells were transfected with siRNAs targeting the indicated genes using the TransIT-TKO® Reagent, and the knockdown percentage was determined using quantitative RT-PCR.