Protocol tips
Upstream tips |
Protocol tips |
Downstream 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 |
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 |
|
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 |
Publication protocol
All siRNAs (Control - 1022076, TLR3 - SI00050043, DDX58 - SI00361809, IFIH1 - SI03648981, RELA - SI00131943 and SI00301672, and RELB - SI00089117 and SI00089131) were purchased from QIAGEN, and nucleofection was performed using AMAXA Biosystems according to the manufacturer's protocol specified as optimal for HT1080 (Solution V with nucleofection mode A-23). The efficiency of each gene-specific siRNA knock-down was calculated based on mRNA levels measured by quantitative real-time PCR whereby the percentage represents the resulting mRNA level of each gene knockdown compared to corresponding control.
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