siRNA / miRNA gene silencing Human - U251 TBX2

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 TBX2 (6909) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	U251 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen). siGENOME Tbx2 siRNAs pool and negative siRNAs pool were obtained from Dharmacon (GE Healthcare, Piscataway, NJ, USA). siRNA was transfected with DharmaFECT1 (GE Healthcare). For each well of a 6-well plate, 0.2 nmol siRNA was used for transfection.

Protocol tips
U251 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen). siGENOME Tbx2 siRNAs pool and negative siRNAs pool were obtained from Dharmacon (GE Healthcare, Piscataway, NJ, USA). siRNA was transfected with DharmaFECT1 (GE Healthcare). For each well of a 6-well plate, 0.2 nmol siRNA was used for transfection.

Protocol tips

U251 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen). siGENOME Tbx2 siRNAs pool and negative siRNAs pool were obtained from Dharmacon (GE Healthcare, Piscataway, NJ, USA). siRNA was transfected with DharmaFECT1 (GE Healthcare). For each well of a 6-well plate, 0.2 nmol siRNA was used for transfection.

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