ON-TARGETplus Human RAB25 (57111) siRNA - SMARTpool

Protocol tips

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.

Publication protocol

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.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing siRNA / miRNA gene silencing Human - ES2 RAB25 using ON-TARGETplus Human RAB25 (57111) siRNA - SMARTpool from Horizon Discovery Ltd..

Manufacturer protocol

Download the product protocol from Horizon Discovery Ltd. for ON-TARGETplus Human RAB25 (57111) siRNA - SMARTpool below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing siRNA / miRNA gene silencing Human - ES2 RAB25 using ON-TARGETplus Human RAB25 (57111) siRNA - SMARTpool from Horizon Discovery Ltd.. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.