Cells were seeded in six‐well plates (NEST Biotechnology Co.LTD., Wuxi, China) at a density of 1 × 105 cells/well and replaced with serum‐free medium when density reached about 80%. 10–20 ul TRAF6 siRNA or negative control siRNA (GenePharma, Shang Hai, China) was mixed with 125 μl opti‐MEM medium (Thermo Fisher Scientific, Waltham, MA, USA). 3.75 μl Lipofectamine‐3000 (Thermo Fisher Scientific) was added to another 125 μl opti‐MEM medium and fully mixed. This was gently mixed and then incubated for 5 min. at room temperature. The untreated groups (without siRNA and transfection agent) were used as blank controls. The mixture was then added to the six‐well plates. Cells were incubated at 37°C for 2–4 days. The transfected cells were then analysed.
Traf6 sirna
Manufactured by GenePharma
Sourced in China
TRAF6 siRNA is a laboratory tool designed to selectively silence the expression of the TRAF6 gene. TRAF6 is an important signaling molecule involved in various cellular processes. This siRNA product can be used to study the functional role of TRAF6 in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
X tremegene sirna transfection reagent, by Roche (2 mentions)
Traf6 sirna, by Roche (2 mentions)
Sirna mate transfection reagent, by GenePharma (2 mentions)
Cd40l, by Thermo Fisher Scientific (2 mentions)
E coli o111 b4, by Merck Group (2 mentions)
Cd40 sirna, by GenePharma (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Six well plate, by NEST Biotechnology (1 mentions)
Entranster r, by Engreen Biosystem (1 mentions)
6 protocols using traf6 sirna
1
siRNA Transfection in Cell Lines
2
miR-146b-5p Mimic and TRAF6 siRNA Transfection
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The dsRNA oligonucleotides of miR-146b-5p mimics, small interfering RNA silencing TRAF6 (TRAF6 siRNA) and scrambled sequence (negative control) were purchased from GenePharma. Their sequences have been listed in Supplementary Table 4 . The U87MG, SNB19 and U251 cells of miR-146b-5p group, TRAF6 siRNA group and negative control group were respectively transfected with the corresponding dsRNA oligonucleotides using X-tremeGENE siRNA Transfection Reagent (Roche). The mock groups of the above cell lines were treated only using the transfection reagent of the same volume.
3
Cellular miRNA and siRNA Transfection
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The dsRNA oligonucleotides of miR-146b-5p mimics, small interfering RNA silencing TRAF6 (TRAF6 siRNA) and scrambled sequence (negative control) were purchased from GenePharma. The U87MG, SNB19 and U251 cells of miR-146b-5p group, TRAF6 siRNA group and negative control group were transfected with the corresponding dsRNA oligonucleotides using X-tremeGENE siRNA Transfection Reagent (Roche). The mock groups were treated with the transfection reagent of the same volume.
4
TRAF6 siRNA Silencing Efficacy
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Prior to transfection, 6×105 cells were seeded in 6-well plates in RPMI-1640 supplemented with 10% FBS at 37°C. The following day, 50 nM of TRAF6 siRNA (Shanghai GenePharma Co, Ltd., Shanghai, China) was added to each well with Entranster™-R (Engreen Biosystem Co., Ltd., Beijing, China) and cultured for a further 48 h at 37°C in an atmosphere containing 5% CO2. RNA interference oligomers were complementary to the TRAF6 mRNA, and were as follows: Sense 5′-GCAGAUGGGGCAUUCAUATT-3′; antisense 5′-UAUGAAUGCCCCAUCUGCTT-3′). The negative control siRNAs were as follows: Sense 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense 5′-ACGUGACACGUUCGGAGAATT-3′. Scrambled siRNAs were used as controls (Shanghai GenePharma Co., Ltd.).
5
Induction of Enteric Glial Cell Sepsis
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To induce the cell sepsis model, EGCs were stimulated by single lipopolysaccharide (LPS) (10 μg/mL, E. coli O111:B4, Sigma-Aldrich, St. Louis, MO, USA), CD40L (1 μg/mL, PeproTech, Rocky Hill, NJ, USA) or LPS + CD40L. After 24 h, the cell culture supernatants were collected as EGCs-conditioned medium for cytokine measurement and Caco-2 cell treatment.
For the siRNA transfection, CD40-siRNA, TRAF6-siRNA, negative control siRNA (NC-siRNA), and siRNA-Mate transfection reagent were purchased from GenePharma Co. Ltd, (Shanghai, China). EGCs were transfected with CD40-siRNA, TRAF6-siRNA or NC-siRNA using siRNA-Mate according to the manufacturer’s protocols for 72 h before LPS and CD40L stimulation.
For the siRNA transfection, CD40-siRNA, TRAF6-siRNA, negative control siRNA (NC-siRNA), and siRNA-Mate transfection reagent were purchased from GenePharma Co. Ltd, (Shanghai, China). EGCs were transfected with CD40-siRNA, TRAF6-siRNA or NC-siRNA using siRNA-Mate according to the manufacturer’s protocols for 72 h before LPS and CD40L stimulation.
6
Induction of Enteric Glial Cell Sepsis
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+ Expand
To induce the cell sepsis model, EGCs were stimulated by single lipopolysaccharide (LPS) (10 μg/mL, E. coli O111:B4, Sigma-Aldrich, St. Louis, MO, USA), CD40L (1 μg/mL, PeproTech, Rocky Hill, NJ, USA) or LPS + CD40L. After 24 h, the cell culture supernatants were collected as EGCs-conditioned medium for cytokine measurement and Caco-2 cell treatment.
For the siRNA transfection, CD40-siRNA, TRAF6-siRNA, negative control siRNA (NC-siRNA), and siRNA-Mate transfection reagent were purchased from GenePharma Co. Ltd, (Shanghai, China). EGCs were transfected with CD40-siRNA, TRAF6-siRNA or NC-siRNA using siRNA-Mate according to the manufacturer’s protocols for 72 h before LPS and CD40L stimulation.
For the siRNA transfection, CD40-siRNA, TRAF6-siRNA, negative control siRNA (NC-siRNA), and siRNA-Mate transfection reagent were purchased from GenePharma Co. Ltd, (Shanghai, China). EGCs were transfected with CD40-siRNA, TRAF6-siRNA or NC-siRNA using siRNA-Mate according to the manufacturer’s protocols for 72 h before LPS and CD40L stimulation.
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