siRNA / miRNA gene silencing Mouse - Neuro 2a TLR4

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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TLR4 siRNA (m)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	N2a cells are a mouse neuroblastoma cell line gifted by Prof. Yu‐Xian Shen (Anhui Medical University, Hefei, Anhui, China). They were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) at 37°C, under a 5% CO2 humidified atmosphere. Ten percent fetal calf serum (HyClone, Marlborough, MA) and 1% penicillin (100 U/mL)‐streptomycin (100 μg/ml) were added to the medium, and the culture medium was changed every 3 days. Sequence‐specific and negative control scrambled siRNAs for mouse TLR4 gene were both purchased from Santa Cruz Biotechnology (catalogs sc‐40261, Santa Cruz Inc). Lipofectamine 2000 was used to transfect N2a cells according to the experimental protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). Briefly, first, N2a cells were incubated at 37°C under 5% CO2 for 24 hours to reach 90% confluence of the monolayer. Then, the medium containing antibiotics was removed, and the cells were washed with phosphate‐buffered saline (PBS) three times. Next, nonantibiotic medium was added. Fourth, Lipofectamine 2000 and siRNA were diluted in 250 μl of Opti‐MEM, followed by incubation at room temperature (RT) for 5 minutes and gentle mixing for 20 minutes. Finally, the mixed solution was added onto the monolayer of N2a cells. Relative experimental assays for mRNA transcription and protein expression were performed at different time points after transfection.

Protocol tips
N2a cells are a mouse neuroblastoma cell line gifted by Prof. Yu‐Xian Shen (Anhui Medical University, Hefei, Anhui, China). They were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) at 37°C, under a 5% CO2 humidified atmosphere. Ten percent fetal calf serum (HyClone, Marlborough, MA) and 1% penicillin (100 U/mL)‐streptomycin (100 μg/ml) were added to the medium, and the culture medium was changed every 3 days. Sequence‐specific and negative control scrambled siRNAs for mouse TLR4 gene were both purchased from Santa Cruz Biotechnology (catalogs sc‐40261, Santa Cruz Inc). Lipofectamine 2000 was used to transfect N2a cells according to the experimental protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). Briefly, first, N2a cells were incubated at 37°C under 5% CO2 for 24 hours to reach 90% confluence of the monolayer. Then, the medium containing antibiotics was removed, and the cells were washed with phosphate‐buffered saline (PBS) three times. Next, nonantibiotic medium was added. Fourth, Lipofectamine 2000 and siRNA were diluted in 250 μl of Opti‐MEM, followed by incubation at room temperature (RT) for 5 minutes and gentle mixing for 20 minutes. Finally, the mixed solution was added onto the monolayer of N2a cells. Relative experimental assays for mRNA transcription and protein expression were performed at different time points after transfection.

Protocol tips

N2a cells are a mouse neuroblastoma cell line gifted by Prof. Yu‐Xian Shen (Anhui Medical University, Hefei, Anhui, China). They were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) at 37°C, under a 5% CO2 humidified atmosphere. Ten percent fetal calf serum (HyClone, Marlborough, MA) and 1% penicillin (100 U/mL)‐streptomycin (100 μg/ml) were added to the medium, and the culture medium was changed every 3 days. Sequence‐specific and negative control scrambled siRNAs for mouse TLR4 gene were both purchased from Santa Cruz Biotechnology (catalogs sc‐40261, Santa Cruz Inc). Lipofectamine 2000 was used to transfect N2a cells according to the experimental protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). Briefly, first, N2a cells were incubated at 37°C under 5% CO2 for 24 hours to reach 90% confluence of the monolayer. Then, the medium containing antibiotics was removed, and the cells were washed with phosphate‐buffered saline (PBS) three times. Next, nonantibiotic medium was added. Fourth, Lipofectamine 2000 and siRNA were diluted in 250 μl of Opti‐MEM, followed by incubation at room temperature (RT) for 5 minutes and gentle mixing for 20 minutes. Finally, the mixed solution was added onto the monolayer of N2a cells. Relative experimental assays for mRNA transcription and protein expression were performed at different time points after transfection.

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