RORC siRNA-NM_011281

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	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.

Publication protocol

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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