siRNA / miRNA gene silencing Mouse - FL83B Rorc

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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Found 1 matching solution for this experiment

RORC siRNA-NM_011281

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Transfection of siRNA targeting ROR‐γ (Sigma, SASI_Mm01_00068648) was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol [Dalby et al., 2004].To establish a stable ROR‐γ knockdown cell line, FL83B cells were infected with a mouse ROR‐γ specific shRNA encoded lentivirus (Sigma). Three coding regions targeting the mouse ROR‐γ starting positions 526 (LV‐shROR‐γ‐1), 1133 (LV‐shROR‐γ‐2), and 1597 (LV‐shROR‐γ‐3) in the sequence (GenBank Acc. No. NM 011281.1) were selected as shRNA target sequences. An shRNA negative control lentiviral particle (LV‐Control) was used as a negative control. To generate a stable cell line, FL83B cells were plated at a density of 1 × 105 cells per 60‐mm culture dish and infected overnight with five multiplicity of infection (MOI) lentiviral particles in the presence of 8 µg/ml hexadimethrine bromide (Sigma). After infection, the transduced cells were selected using 10 µg/ml puromycin (Sigma) for 2 weeks and incubated at 37°C in a humidified incubator with 5% CO2. Suppression of ROR‐γ expression in selected cells was confirmed by Western blot analysis.

Protocol tips
Transfection of siRNA targeting ROR‐γ (Sigma, SASI_Mm01_00068648) was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol [Dalby et al., 2004].To establish a stable ROR‐γ knockdown cell line, FL83B cells were infected with a mouse ROR‐γ specific shRNA encoded lentivirus (Sigma). Three coding regions targeting the mouse ROR‐γ starting positions 526 (LV‐shROR‐γ‐1), 1133 (LV‐shROR‐γ‐2), and 1597 (LV‐shROR‐γ‐3) in the sequence (GenBank Acc. No. NM 011281.1) were selected as shRNA target sequences. An shRNA negative control lentiviral particle (LV‐Control) was used as a negative control. To generate a stable cell line, FL83B cells were plated at a density of 1 × 105 cells per 60‐mm culture dish and infected overnight with five multiplicity of infection (MOI) lentiviral particles in the presence of 8 µg/ml hexadimethrine bromide (Sigma). After infection, the transduced cells were selected using 10 µg/ml puromycin (Sigma) for 2 weeks and incubated at 37°C in a humidified incubator with 5% CO2. Suppression of ROR‐γ expression in selected cells was confirmed by Western blot analysis.

Protocol tips

Transfection of siRNA targeting ROR‐γ (Sigma, SASI_Mm01_00068648) was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol [Dalby et al., 2004].To establish a stable ROR‐γ knockdown cell line, FL83B cells were infected with a mouse ROR‐γ specific shRNA encoded lentivirus (Sigma). Three coding regions targeting the mouse ROR‐γ starting positions 526 (LV‐shROR‐γ‐1), 1133 (LV‐shROR‐γ‐2), and 1597 (LV‐shROR‐γ‐3) in the sequence (GenBank Acc. No. NM 011281.1) were selected as shRNA target sequences. An shRNA negative control lentiviral particle (LV‐Control) was used as a negative control. To generate a stable cell line, FL83B cells were plated at a density of 1 × 105 cells per 60‐mm culture dish and infected overnight with five multiplicity of infection (MOI) lentiviral particles in the presence of 8 µg/ml hexadimethrine bromide (Sigma). After infection, the transduced cells were selected using 10 µg/ml puromycin (Sigma) for 2 weeks and incubated at 37°C in a humidified incubator with 5% CO2. Suppression of ROR‐γ expression in selected cells was confirmed by Western blot analysis.

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