siRNA / miRNA gene silencing Rat - H9c2 NF-κB RelA (p65)

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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Myocardiac H9c2 cell (Catalog. No. CRL-1446), a clonal line derived from embryonic rat heart, was from American Type Culture Collection (ATCC, Manassas, VA). The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma Co., St. Louis, MI) with Dglucose at 4.5 g/L, 10% foetal bovine serum (FBS), 10,000 U/L penicillin, and 10 mg/L streptomycin, in an incubator with 5% CO2, at 37 °C and the medium was changed every 2 days. When confluence reached the cells (between passages 3 and 5) they were subcultured by detaching with 0.25% trypsin–EDTA solution (Sigma Co.) and re-seeding into new plates at a ratio of 1:5 in DMEM with 10% steroid-free FBS (Sigma Co.). Cells at ~75% confluence were cultured in serum-free DMEM, treated with the hormone and its receptor blocker (see below), and subjected to hydrogen peroxide (H2O2, 200 μM, 6 h) treatment.Expression of nuclear NF-κB RelA (p65) in H9c2 myocytes was knocked down by RNA interference using a RelA/p65 siRNA Transfection Kit (GeneResearch Co. Australia) that contained SignalSilence® NF-κB p65 siRNA I (2 μM, mouse specific) and Thermo Scientific DharmaFECT® 1 siRNA transfection reagent. As testing in cultured H9c2 cells by the manufacturer, siRNA (100 nM) complexed with the reagent resulted in silencing (reduction) of mRNA (in QuantiGene® branched DNA/RNA assay) at N90% with little toxicity (cell viability N95% in the alamarBlue® assay). In this study siRNA transfection was performed in accordance with the manufacturer's protocol optimised for use with H9c2 cells in culture. In brief, cells were washed with serum-free DMEM, incubated with the transfection reagent (7 μL in 343 μL DMEM) containing RelA/p65 siRNA (100 nM) for 2–4 h, and continued to culture in serum (10%, steroid-free)-enriched DMEM with the siRNA for 48 h before being ready for the hormonal testing.
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