siRNA / miRNA gene silencing Human - A431 RCP/RAB11FIP1

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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A431 cells were all purchased from the ATCC and cultured in DMEM containing 10% FBS, 1% penicillin/streptomycin, and 1% glutamine at 37 °C and 5% CO2. Knockdown in A431 cells were achieved by transfection of siRNA using the AMAXA nucleofection method, solution V, and protocols X-001. The following siRNA oligos were used: control siRNA 1, 5’-GCAACGGCAUUCCACCUUU(TT)-3’; control siRNA 2 control pool of four siRNAs (Dharmacon, catalog no. D001810-1020); RCP (SMARTpool of four siRNAs (Dharmacon, catalog no. L-015968-00-0005) was used. All siRNA oligos were used as a combination of two or four siRNAs, and each individual siRNA was tested for efficiency of knockdown and off-target effects.
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