siRNA / miRNA gene silencing Human - HT-1080 TLR3

Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of cells.

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Upstream tips
Pre-warm the supplemented Nucleofector Solution

pre-incubate plates filled with 1.0 ml of culture medium containing serum and supplements in a humidified 37°C/5% CO2 incubator
Protocol tips
seed 1x10^6 – 5x10^6 cells in the wells containing above medium and centrifuge at 90xg for 10 min.

Resuspend pellet and add Nucleofector Solution to a final concentration of 1x106 – 5x106
cells/100 μl

Mix 100 μl of cell suspension with siRNA
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