siRNA / miRNA gene silencing Rat - UMR‐106 Atg7

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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The rat UMR-106 osteosarcoma cell line75 was maintained in Dulbecco modified Eagle's medium (Lonza, BE12-604) supplemented with 10% Hyclone fetal calf serum (Thermo Scientific, SH30071.03). The UMR-106 GFP-LC3 clone 4 cells correspond to the UMR-106 cell line stably transfected with the pGFP-LC3 construct. These cells are maintained in the same medium in the presence of G418 (800 μg/ml; Sigma-Aldrich, G8168). For mineralization, the cells were cultured in α-MEM (Lonza, BE02-002) supplemented with 10% Hyclone fetal calf serum, CaCl2 (1.4 mM; Merck 2382), ascorbic acid (50 μg/mL; Sigma-Aldrich A4034) and dexamethasone (20 μg/mL; Sigma-Aldrich, D8893) for 3 d, and then for 3 d in the same medium in the presence of β-glycerophosphate (50 mg/mL; Sigma-Aldrich G9891). ON-TARGET plus SMARTpool siRNA (Dharmacon RNAi Technologies, Abgene Ltd) for rat Atg7 (L095596-01) and Becn1 (L-099237-01) were transfected in the UMR-106 cell line using nucleofection and Amaxa solution V (Lonza, VCA-1003).
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