Rn_Tlr3_1 FlexiTube siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 2×10^4 cells	siRNA concentration-10 nM Incubate HiPerFect Transfection Reagent to diluted siRNA for 10–15 min at RT. 24 h post transfection treat cells with infected with 0.1 MOI of JEV or 1 μg/μLof TLR3 ligand poly I:C(positive control) and incubate for 1h and replace medium. Analyse cells after 18h

Upstream tips
Seed 2×10^4 cells
Protocol tips
siRNA concentration-10 nM Incubate HiPerFect Transfection Reagent to diluted siRNA for 10–15 min at RT. 24 h post transfection treat cells with infected with 0.1 MOI of JEV or 1 μg/μLof TLR3 ligand poly I:C(positive control) and incubate for 1h and replace medium. Analyse cells after 18h

Publication protocol

Neuro2a cells were transfected with TLR3 siRNA specific for mouse (Genesolution siRNA, Qiagen) using a commercially obtained HiPerFect Transfection Reagent (Qiagen) as per manufacturer's instructions. Briefly, 2×104 Neuro2a cells were seeded per well of a 24-well plate in 1 mL/well of DMEM containing serum and antibiotics and incubated at 37°C in a CO2 incubator until 80% confluence of the monolayer was attained. For transfection, 1 μL (10 nM) of each of four siRNA reagents and 6 μL of HiPerFect Transfection reagent were mixed in 90 μL of DMEM to obtain a volume of 100 μL/per transfection, vortexed, and incubated at room temperature for 10–15 min to form transfection complexes. At the end of incubation, 100 μL of transfection mix was added dropwise to the wells of the plate with gentle swirling of the plate to ensure uniform distribution of complexes. The cells were subsequently incubated under their normal growth conditions. At the end of 24 h, the transfection complexes were removed and the cells were either infected with 0.1 MOI of JEV or 1 μg/μLof TLR3 ligand poly I:C(positive control) and incubated at 37°C for 1 h. At the end of incubation, the virus /poly I:C was removed and fresh maintenance medium was added to the cells and incubated for a further period of 18 h. Cells were subsequently harvested and RNA extracted from infected as well as siRNA treated and untreated cells by Tri-reagent method as described above. The RNA was converted to cDNA and real-time PCR was carried out for presence of JEV-NS5 protein gene and TLR3 as described.

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Manufacturer protocol

Download the product protocol from Qiagen for Rn_Tlr3_1 FlexiTube siRNA below.

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