siRNA / miRNA gene silencing Human - PANC-1 GSK-3α

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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Found 1 matching solution for this experiment

siGENOME Human GSK3A (2931) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Panc-1 (CRL-1496) and pancreatic cancer cell lines was obtained from the American Type Culture Collection (Manassas, VA). Panc-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. Cells were cultured in DMEM supplemented with 0.5% fetal bovine serum for 24 hour prior to experimentation. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). The following siRNA (siGenome SMARTpool) was obtained from Dharmacon (Layfayette, CO) as a pool of four annealed double-stranded RNA oligonucleotides: GSK-3α (M-003009-01), and non-targeting control #3 (D001201-03). In brief, cells were cultured to 70% confluency in six-well plates. Dharmafect 1 transfection reagent (Layfayette, CO) was used to transfect 100nM siRNA according to manufacturer’s instruction.

Protocol tips
Panc-1 (CRL-1496) and pancreatic cancer cell lines was obtained from the American Type Culture Collection (Manassas, VA). Panc-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. Cells were cultured in DMEM supplemented with 0.5% fetal bovine serum for 24 hour prior to experimentation. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). The following siRNA (siGenome SMARTpool) was obtained from Dharmacon (Layfayette, CO) as a pool of four annealed double-stranded RNA oligonucleotides: GSK-3α (M-003009-01), and non-targeting control #3 (D001201-03). In brief, cells were cultured to 70% confluency in six-well plates. Dharmafect 1 transfection reagent (Layfayette, CO) was used to transfect 100nM siRNA according to manufacturer’s instruction.

Protocol tips

Panc-1 (CRL-1496) and pancreatic cancer cell lines was obtained from the American Type Culture Collection (Manassas, VA). Panc-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. Cells were cultured in DMEM supplemented with 0.5% fetal bovine serum for 24 hour prior to experimentation. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). The following siRNA (siGenome SMARTpool) was obtained from Dharmacon (Layfayette, CO) as a pool of four annealed double-stranded RNA oligonucleotides: GSK-3α (M-003009-01), and non-targeting control #3 (D001201-03). In brief, cells were cultured to 70% confluency in six-well plates. Dharmafect 1 transfection reagent (Layfayette, CO) was used to transfect 100nM siRNA according to manufacturer’s instruction.

Manufacturer protocol Publication protocol 1 Relevant paper

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