ON-TARGETplus Human MDK (4192) siRNA - SMARTpool

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	Human LNCaP cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured in high glucose DMEM/Hams’ F-12 medium (50/50 mix) (Winsent, Quebec, Canada) with 2 mM L-glutamine, 1500 mg/l sodium bicarbonate and 10% fetal bovine serum (FBS; Life Technologies, USA). To improve the adherence of LNCaP cells to the culture surface, the flask surface was coated with poly-l-lysine (Sigma, St. Louis, Mo., USA . LNCaP cells were treated with 10 nM 5α-dihydrotestosterone (DHT) (Sigma, St. Louis, Mo., USA). The cells were maintained at 37 °C and 5% CO2. LNCaP cells were plated onto six-well plates at a density of 2.5 × 105 cells in DMEM/F12 medium without FBS and antibiotics. Following 16 h of incubation, cells were transiently transfected with midkine ON-TARGETplus human SMARTpool siRNA (catalog # L-003677-00, Dharmacon, Inc., Lafayette, CO, USA) with 2 μl/ml of Dharmafect-2 reagent (Dharmacon, Inc) according to the manufacturer’s instructions. The final concentration of siRNA was 12.5, 25 and 50 pmol. The plates were incubated for 6 h after transfection, and then media was changed with fresh DMEM/ F12 containing 10% FBS. Based on our results 50 pmol siRNA transfection was selected for further studies.

Protocol tips

Human LNCaP cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured in high glucose DMEM/Hams’ F-12 medium (50/50 mix) (Winsent, Quebec, Canada) with 2 mM L-glutamine, 1500 mg/l sodium bicarbonate and 10% fetal bovine serum (FBS; Life Technologies, USA). To improve the adherence of LNCaP cells to the culture surface, the flask surface was coated with poly-l-lysine (Sigma, St. Louis, Mo., USA . LNCaP cells were treated with 10 nM 5α-dihydrotestosterone (DHT) (Sigma, St. Louis, Mo., USA). The cells were maintained at 37 °C and 5% CO2. LNCaP cells were plated onto six-well plates at a density of 2.5 × 105 cells in DMEM/F12 medium without FBS and antibiotics. Following 16 h of incubation, cells were transiently transfected with midkine ON-TARGETplus human SMARTpool siRNA (catalog # L-003677-00, Dharmacon, Inc., Lafayette, CO, USA) with 2 μl/ml of Dharmafect-2 reagent (Dharmacon, Inc) according to the manufacturer’s instructions. The final concentration of siRNA was 12.5, 25 and 50 pmol. The plates were incubated for 6 h after transfection, and then media was changed with fresh DMEM/ F12 containing 10% FBS. Based on our results 50 pmol siRNA transfection was selected for further studies.

Publication protocol

Human LNCaP cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured in high glucose DMEM/Hams’ F-12 medium (50/50 mix) (Winsent, Quebec, Canada) with 2 mM L-glutamine, 1500 mg/l sodium bicarbonate and 10% fetal bovine serum (FBS; Life Technologies, USA). To improve the adherence of LNCaP cells to the culture surface, the flask surface was coated with poly-l-lysine (Sigma, St. Louis, Mo., USA . LNCaP cells were treated with 10 nM 5α-dihydrotestosterone (DHT) (Sigma, St. Louis, Mo., USA). The cells were maintained at 37 °C and 5% CO2. LNCaP cells were plated onto six-well plates at a density of 2.5 × 105 cells in DMEM/F12 medium without FBS and antibiotics. Following 16 h of incubation, cells were transiently transfected with midkine ON-TARGETplus human SMARTpool siRNA (catalog # L-003677-00, Dharmacon, Inc., Lafayette, CO, USA) with 2 μl/ml of Dharmafect-2 reagent (Dharmacon, Inc) according to the manufacturer’s instructions. The final concentration of siRNA was 12.5, 25 and 50 pmol. The plates were incubated for 6 h after transfection, and then media was changed with fresh DMEM/ F12 containing 10% FBS. Based on our results 50 pmol siRNA transfection was selected for further studies.

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