siRNA / miRNA gene silencing Human - LNCap MDK

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.

1 Matching solution
Start a discussion

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 1 matching solution for this experiment

ON-TARGETplus Human MDK (4192) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream 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.

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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment