siRNA / miRNA gene silencing Human - ES2 RAB25

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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ON-TARGETplus Human RAB25 (57111) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	ES-2 human ovarian cancer cell lines (donated by the University of Texas, M. D. Anderson Cancer Center, Houston, TX, USA) were grown in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% FBS (Hyclone, Logan, UT, USA) in a humidified atmosphere of 5% CO2 at 37°C. These cells were sub-cultured by adding 0.05% trypsin-0.01% EDTA (Gibco) when the cells reached 80% confluence. For experiments involving the pharmacological inhibitor, the cells were serum-starved for 12 h and then treated with U0126 (Sigma Aldrich, St. Louis, MO, USA) at a concentration of 10 μM for 24 h. Cells treated with DMSO (Sigma Aldrich) served as the control.The RAB25 ON-TARGET plus SMART pool siRNA (siRab) and siGLO non-targeting siCONTROL siRNA (siNon) were purchased from Dharmacon (Lafayette, CO, USA). Cells were transfected with siRab or siNon using DharmaFCET 1 reagent (Dharmacon) according to the manufacturer’s instructions. Briefly, the siRNA and transfection reagent were diluted in serum-free Opti-MEM and mixed. Following incubation at room temperature for 20 min, the mixture was added to the cells at a final siRNA concentration of 50 nM. Following incubation for 6 h, FBS was added to achieve a final concentration of 10% and the cells were incubated for 24 h prior to subsequent treatment. Cells treated with DharmaFECT 1 reagent served as the control.

Protocol tips
ES-2 human ovarian cancer cell lines (donated by the University of Texas, M. D. Anderson Cancer Center, Houston, TX, USA) were grown in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% FBS (Hyclone, Logan, UT, USA) in a humidified atmosphere of 5% CO2 at 37°C. These cells were sub-cultured by adding 0.05% trypsin-0.01% EDTA (Gibco) when the cells reached 80% confluence. For experiments involving the pharmacological inhibitor, the cells were serum-starved for 12 h and then treated with U0126 (Sigma Aldrich, St. Louis, MO, USA) at a concentration of 10 μM for 24 h. Cells treated with DMSO (Sigma Aldrich) served as the control.The RAB25 ON-TARGET plus SMART pool siRNA (siRab) and siGLO non-targeting siCONTROL siRNA (siNon) were purchased from Dharmacon (Lafayette, CO, USA). Cells were transfected with siRab or siNon using DharmaFCET 1 reagent (Dharmacon) according to the manufacturer’s instructions. Briefly, the siRNA and transfection reagent were diluted in serum-free Opti-MEM and mixed. Following incubation at room temperature for 20 min, the mixture was added to the cells at a final siRNA concentration of 50 nM. Following incubation for 6 h, FBS was added to achieve a final concentration of 10% and the cells were incubated for 24 h prior to subsequent treatment. Cells treated with DharmaFECT 1 reagent served as the control.

Protocol tips

ES-2 human ovarian cancer cell lines (donated by the University of Texas, M. D. Anderson Cancer Center, Houston, TX, USA) were grown in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% FBS (Hyclone, Logan, UT, USA) in a humidified atmosphere of 5% CO2 at 37°C. These cells were sub-cultured by adding 0.05% trypsin-0.01% EDTA (Gibco) when the cells reached 80% confluence. For experiments involving the pharmacological inhibitor, the cells were serum-starved for 12 h and then treated with U0126 (Sigma Aldrich, St. Louis, MO, USA) at a concentration of 10 μM for 24 h. Cells treated with DMSO (Sigma Aldrich) served as the control.The RAB25 ON-TARGET plus SMART pool siRNA (siRab) and siGLO non-targeting siCONTROL siRNA (siNon) were purchased from Dharmacon (Lafayette, CO, USA). Cells were transfected with siRab or siNon using DharmaFCET 1 reagent (Dharmacon) according to the manufacturer’s instructions. Briefly, the siRNA and transfection reagent were diluted in serum-free Opti-MEM and mixed. Following incubation at room temperature for 20 min, the mixture was added to the cells at a final siRNA concentration of 50 nM. Following incubation for 6 h, FBS was added to achieve a final concentration of 10% and the cells were incubated for 24 h prior to subsequent treatment. Cells treated with DharmaFECT 1 reagent served as the control.

Manufacturer protocol Publication protocol 1 Relevant paper

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