- Broad Spectrum siRNA and Plasmid DNA Delivery – Utilize one transfection reagent and protocol for a wide variety of cells
- Low Cellular Toxicity – Maintain cell density and reduce experimental biases
- Reproducible Results – Obtain consistent, targeted gene knockdowns from day to day
- High Knockdown Efficiency – Achieve optimal gene silencing in a large percentage of cells to ensure experimental success
Stem Cell Applications
Optimized transfection protocol for iCell® Cardiomyocytes (Cellular Dynamics International) using TransIT-TKO® Transfection Reagent: PROTOCOL (PDF) | iCell® Page
Cell Types successfully transfected at Mirus using TransIT-TKO® Transfection Reagent (PDF)
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 µl 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.
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.
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.
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.
|Cell Line (Source)||Endogenous Transcript||Knockdown Efficiency|
|BNL CL.2 (mouse liver)||MAPK1||80%|
|HeLa (human cervix)||Lamin A/C||80%|
|Hepa1c1c7 (mouse liver)||MAPK1||80%|
|HepG2 (human liver)||MAPK1||80%|
|Secondary Human Astrocytes||Lamin A/C||80%|
|Primary Mouse Hepatocytes||ABC A1||70%|
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.