siRNA / miRNA gene silencing Human - HT-1376 GLUT1

Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of cells.

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Glut1 siRNA and shRNA Plasmids (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Human bladder cancer cell lines UM-UC-3 and HT-1376 derived from high-grade transitional cell carcinoma (from the American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% (v/v) of fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Sigma). GLUT1 was depleted in human bladder cancer cells using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). HT-1376 bladder cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with GLUT1. Cells were also transfected with a scrambled siRNA in parallel as controls.For each transfection, cells were treated for 5 h with 2.4 µM of siRNA in transfection medium (Santa Cruz) containing 0.5 µL/cm2 of transfection reagent (Santa Cruz). After incubation, complete media was added and the cells were incubated for 24 or 48 h. GLUT1 downregulation was evaluated 24 h or 48 h post-transfection by Western blotting. The uptake and PDT experiments were performed 24 h or 48 h post-transfection with GLUT1 hsiRNA or galectin-1 hsiRNA, respectively.

Protocol tips
Human bladder cancer cell lines UM-UC-3 and HT-1376 derived from high-grade transitional cell carcinoma (from the American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% (v/v) of fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Sigma). GLUT1 was depleted in human bladder cancer cells using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). HT-1376 bladder cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with GLUT1. Cells were also transfected with a scrambled siRNA in parallel as controls.For each transfection, cells were treated for 5 h with 2.4 µM of siRNA in transfection medium (Santa Cruz) containing 0.5 µL/cm2 of transfection reagent (Santa Cruz). After incubation, complete media was added and the cells were incubated for 24 or 48 h. GLUT1 downregulation was evaluated 24 h or 48 h post-transfection by Western blotting. The uptake and PDT experiments were performed 24 h or 48 h post-transfection with GLUT1 hsiRNA or galectin-1 hsiRNA, respectively.

Protocol tips

Human bladder cancer cell lines UM-UC-3 and HT-1376 derived from high-grade transitional cell carcinoma (from the American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% (v/v) of fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Sigma). GLUT1 was depleted in human bladder cancer cells using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). HT-1376 bladder cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with GLUT1. Cells were also transfected with a scrambled siRNA in parallel as controls.For each transfection, cells were treated for 5 h with 2.4 µM of siRNA in transfection medium (Santa Cruz) containing 0.5 µL/cm2 of transfection reagent (Santa Cruz). After incubation, complete media was added and the cells were incubated for 24 or 48 h. GLUT1 downregulation was evaluated 24 h or 48 h post-transfection by Western blotting. The uptake and PDT experiments were performed 24 h or 48 h post-transfection with GLUT1 hsiRNA or galectin-1 hsiRNA, respectively.

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