siRNA / miRNA gene silencing Human - HepG2 FGB

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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Silencer select_FGB siRNA

Thermo Fisher Scientific

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	A human hepatocyte cell line, HepG2 cells, was cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Inc Logan, UT, USA), 50 U/ml penicillin, and 50 μg/ml streptomycin (GIBCO) in a 5% CO2 incubator at 37 °C. Silencer Select Pre-designed FGB siRNA (catalog no. 4392420, ID s5119), and Negative Control #1 siRNA (catalog no. 4390843) were obtained from Ambion (Austin, TX, USA). Each siRNA was reversely transfected into HepG2 cells in technical triplicates in 3 independent experiments. Briefly, each siRNA was mixed with 100 μl OPTI-MEM I Reduced Serum Medium (GIBCO) in a 24-well tissue culture plate, and 1 μl Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) was added to each well, mixed gently, and incubated for 20 minutes at room temperature according to the manufacturer's protocol. Then, 500 μl HepG2 cells suspension containing 5×104 cells was added to the mixture of siRNA reagent. The final concentrations of FGA, FGB, and FGG siRNA varied between 1.0 and 30 nM, negative control siRNA was used at 10 nM, and the absence of siRNA was used as a non-transfected control. To obtain a nearly 50% decrease of FGA, FGB, or FGG mRNA expression in each experiment, siRNA concentrations varied from 1.0 to 2.0 nM.

Protocol tips
A human hepatocyte cell line, HepG2 cells, was cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Inc Logan, UT, USA), 50 U/ml penicillin, and 50 μg/ml streptomycin (GIBCO) in a 5% CO2 incubator at 37 °C. Silencer Select Pre-designed FGB siRNA (catalog no. 4392420, ID s5119), and Negative Control #1 siRNA (catalog no. 4390843) were obtained from Ambion (Austin, TX, USA). Each siRNA was reversely transfected into HepG2 cells in technical triplicates in 3 independent experiments. Briefly, each siRNA was mixed with 100 μl OPTI-MEM I Reduced Serum Medium (GIBCO) in a 24-well tissue culture plate, and 1 μl Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) was added to each well, mixed gently, and incubated for 20 minutes at room temperature according to the manufacturer's protocol. Then, 500 μl HepG2 cells suspension containing 5×104 cells was added to the mixture of siRNA reagent. The final concentrations of FGA, FGB, and FGG siRNA varied between 1.0 and 30 nM, negative control siRNA was used at 10 nM, and the absence of siRNA was used as a non-transfected control. To obtain a nearly 50% decrease of FGA, FGB, or FGG mRNA expression in each experiment, siRNA concentrations varied from 1.0 to 2.0 nM.

Protocol tips

A human hepatocyte cell line, HepG2 cells, was cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Inc Logan, UT, USA), 50 U/ml penicillin, and 50 μg/ml streptomycin (GIBCO) in a 5% CO2 incubator at 37 °C. Silencer Select Pre-designed FGB siRNA (catalog no. 4392420, ID s5119), and Negative Control #1 siRNA (catalog no. 4390843) were obtained from Ambion (Austin, TX, USA). Each siRNA was reversely transfected into HepG2 cells in technical triplicates in 3 independent experiments. Briefly, each siRNA was mixed with 100 μl OPTI-MEM I Reduced Serum Medium (GIBCO) in a 24-well tissue culture plate, and 1 μl Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) was added to each well, mixed gently, and incubated for 20 minutes at room temperature according to the manufacturer's protocol. Then, 500 μl HepG2 cells suspension containing 5×104 cells was added to the mixture of siRNA reagent. The final concentrations of FGA, FGB, and FGG siRNA varied between 1.0 and 30 nM, negative control siRNA was used at 10 nM, and the absence of siRNA was used as a non-transfected control. To obtain a nearly 50% decrease of FGA, FGB, or FGG mRNA expression in each experiment, siRNA concentrations varied from 1.0 to 2.0 nM.

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