siRNA / miRNA gene silencing Human - A549 PCSK9

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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PCSK9 siRNA (h)

Santa Cruz Biotechnology

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	The A549 human LAD cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were routinely grown in RPMI-1640 medium (Hyclone; GE Healthcare, Chalfont, UK) containing 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin (Sigma-Aldrich, Merck-Millipore, Darmstadt, Germany) and 100 µg/ml streptomycin (Sigma-Aldrich) at 37°C in a humidified atmosphere containing 5% CO2. The medium was replaced every 2–3 days and upon reaching 80% confluence, they were passaged at a 1:2 ratio. A549 cells were transfected with 100 nM PCSK9 siRNA (cat. no. sc-45482; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; Genbank ID for PCSK9: NM_174936) or control siRNA (scrambled siRNA, a universal negative control; cat. no. sc-37007; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) with GeneSilencer siRNA transfection reagent (Genlantis, San Diego, CA, USA), according to the manufacturer's instructions. At 48 h after transfection, the efficiency of siRNA-mediated PCSK9 knockdown was determined by western blot analysis.

Protocol tips
The A549 human LAD cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were routinely grown in RPMI-1640 medium (Hyclone; GE Healthcare, Chalfont, UK) containing 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin (Sigma-Aldrich, Merck-Millipore, Darmstadt, Germany) and 100 µg/ml streptomycin (Sigma-Aldrich) at 37°C in a humidified atmosphere containing 5% CO2. The medium was replaced every 2–3 days and upon reaching 80% confluence, they were passaged at a 1:2 ratio. A549 cells were transfected with 100 nM PCSK9 siRNA (cat. no. sc-45482; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; Genbank ID for PCSK9: NM_174936) or control siRNA (scrambled siRNA, a universal negative control; cat. no. sc-37007; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) with GeneSilencer siRNA transfection reagent (Genlantis, San Diego, CA, USA), according to the manufacturer's instructions. At 48 h after transfection, the efficiency of siRNA-mediated PCSK9 knockdown was determined by western blot analysis.

Protocol tips

The A549 human LAD cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were routinely grown in RPMI-1640 medium (Hyclone; GE Healthcare, Chalfont, UK) containing 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin (Sigma-Aldrich, Merck-Millipore, Darmstadt, Germany) and 100 µg/ml streptomycin (Sigma-Aldrich) at 37°C in a humidified atmosphere containing 5% CO2. The medium was replaced every 2–3 days and upon reaching 80% confluence, they were passaged at a 1:2 ratio. A549 cells were transfected with 100 nM PCSK9 siRNA (cat. no. sc-45482; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; Genbank ID for PCSK9: NM_174936) or control siRNA (scrambled siRNA, a universal negative control; cat. no. sc-37007; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) with GeneSilencer siRNA transfection reagent (Genlantis, San Diego, CA, USA), according to the manufacturer's instructions. At 48 h after transfection, the efficiency of siRNA-mediated PCSK9 knockdown was determined by western blot analysis.

Manufacturer protocol Publication protocol 1 Relevant paper

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