siRNA / miRNA gene silencing Rat - NPC Lrp6

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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NPCs were transfected with plasmid expressing GFP, tPAGFP (kindly donated by Dr. Bethe Scallatter), or siRNA constructs against tPA, β-catenin, and LRP 5/6 using Lipofectamine 2000 (Invitrogen). Rat tPA-specific siRNA Stealth RNAi™ siRNA Select RNAi was purchased from Invitrogen (cat. no. RSS304050). LRP5- and LRP6-specific siRNA siGENOMESMARTpool Rat LOC312781 and LOC293649 were purchased from Dharmacon. β-Catenin siRNA was purchased from Invitrogen (cat. no. RSS331356). As negative controls, NPCs were transfected with control siRNAs containing the same content of GC as target siRNAs. NPCs were plated at a density of 200,000 cells/cm2 on a plate. Transfection conditions such as transfection time and concentration of DNA/RNA were adjusted according to the manufacturer's guide for LipofectamineTM 2000. The cells were harvested with RIPA buffer 24 h or 3 days after transfection or fixed with 4 % PFA for further experiment
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