STEAP1 siRNA

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	The LNCaP prostate cancer cell line was purchased from the European Collection of Cell Cultures (ECACC, UK) and maintained in RPMI 1640 medium (Gibco, Scotland) supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Germany) and 1% penicillin/streptomycin (Invitrogen, USA), in a humidifed chamber at 37 °C and a 5% CO2 atmosphere. LNCaP cells at 40% confuence in six-plate multiwells were transfected with 50 and 100 nM of a small interfering RNA (siRNA) targeting the STEAP1 (s25634) (Ambion, USA) and 5 µL of Lipofectamine 2000 (Invitrogen, USA) for 24 h in Opti-MEM medium (Invitrogen, USA), as recommended by the manufacturer. As a control for STEAP1-specifc targeting, a scrambled siRNA sequence (AM4635) was used. The efciency of STEAP1 knockdown expression was analyzed by quantitative real-time PCR (qPCR). In order to confrm the STEAP1 knockdown at the protein level, LNCaP cells were transfected with 50 nM of siRNA for 24 h, after which the medium was replaced to complete medium. Cells were harvested at 0, 12, 24 and 48 h after transfection, and STEAP1 protein expression was analyzed by Western blot.

Protocol tips

The LNCaP prostate cancer cell line was purchased from the European Collection of Cell Cultures (ECACC, UK) and maintained in RPMI 1640 medium (Gibco, Scotland) supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Germany) and 1% penicillin/streptomycin (Invitrogen, USA), in a humidifed chamber at 37 °C and a 5% CO2 atmosphere. LNCaP cells at 40% confuence in six-plate multiwells were transfected with 50 and 100 nM of a small interfering RNA (siRNA) targeting the STEAP1 (s25634) (Ambion, USA) and 5 µL of Lipofectamine 2000 (Invitrogen, USA) for 24 h in Opti-MEM medium (Invitrogen, USA), as recommended by the manufacturer. As a control for STEAP1-specifc targeting, a scrambled siRNA sequence (AM4635) was used. The efciency of STEAP1 knockdown expression was analyzed by quantitative real-time PCR (qPCR). In order to confrm the STEAP1 knockdown at the protein level, LNCaP cells were transfected with 50 nM of siRNA for 24 h, after which the medium was replaced to complete medium. Cells were harvested at 0, 12, 24 and 48 h after transfection, and STEAP1 protein expression was analyzed by Western blot.

Publication protocol

The LNCaP prostate cancer cell line was purchased from the European Collection of Cell Cultures (ECACC, UK) and maintained in RPMI 1640 medium (Gibco, Scotland) supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Germany) and 1% penicillin/streptomycin (Invitrogen, USA), in a humidifed chamber at 37 °C and a 5% CO2 atmosphere. LNCaP cells at 40% confuence in six-plate multiwells were transfected with 50 and 100 nM of a small interfering RNA (siRNA) targeting the STEAP1 (s25634) (Ambion, USA) and 5 µL of Lipofectamine 2000 (Invitrogen, USA) for 24 h in Opti-MEM medium (Invitrogen, USA), as recommended by the manufacturer. As a control for STEAP1-specifc targeting, a scrambled siRNA sequence (AM4635) was used. The efciency of STEAP1 knockdown expression was analyzed by quantitative real-time PCR (qPCR). In order to confrm the STEAP1 knockdown at the protein level, LNCaP cells were transfected with 50 nM of siRNA for 24 h, after which the medium was replaced to complete medium. Cells were harvested at 0, 12, 24 and 48 h after transfection, and STEAP1 protein expression was analyzed by Western blot.

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