siRNA / miRNA gene silencing Human - LNCap STEAP1

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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STEAP1 siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream 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.

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.

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.

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