TransIT-TKO¢ç Transfection ReagentA high efficiency, low toxicity, siRNA transfection reagent for mammalian cells
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TransIT-TKOⓇ°ú TransIT-siQUESTⓇ small RNA (siRNA and duplex miRNA) Transfection ½Ã¾àÀº ±¤¹üÀ§ÇÏ°Ô »ç¿ëµÇ´Â Á¦Ç°À̸ç ÃÖ¼ÒÀÇ ¼¼Æ÷ µ¶¼ºÀ» °¡Áö°í ÀÖ½À´Ï´Ù. ÀÌ µÎ ½Ã¾àÀº »ç¿ëÀÚ°¡ ÀÚ½ÅÀÇ Æ¯Á¤ÇÑ ¼¼Æ÷ÁÖ¿¡ ÀûÇÕÇÑ ÃÖ»óÀÇ transfection ½Ã¾àÀ» Á¶¼º¿¡ µû¶ó ¼±Åà ÇÏ¿© »ç¿ëÇÒ ¼ö ÀÖ½À´Ï´Ù. Figure 1. High Efficiency Endogenous Knockdown in iCell¢ç Cardiomyocytes. The TransIT-TKO¢ç 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¢ç (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.
Figure 2. Delivery of Fluorescently-Labeled siRNA using TransIT-TKO¢ç Transfection Reagent. HeLa (70% confluence) cells in 12-well plates were transfected with TransIT-TKO¢ç Transfection Reagent (3 ¥ìl/well) and Label IT¢ç 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¢ç-3 (nuclei, BLUE) (Life Technologies) and Alexa Fluor¢ç 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¢ç Reagent in HeLa cells. Firefly and sea pansy luciferase reporter vectors were co-transfected into HeLa cells using TransIT¢ç-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¢ç Reagent. Primary mouse hepatocytes were transfected with the indicated siRNAs or a non-targeting control siRNA using the TransIT-TKO¢ç 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¢ç Reagent. Various cells were transfected with siRNAs targeting the indicated genes using the TransIT-TKO¢ç Reagent, and the knockdown percentage was determined using quantitative RT-PCR.
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