Seeded onto 12 mm glass coverslips (Electron Microscopy Sciences) were 4 × 105 HEK-293T cells. After 16 to 20 h, 80 µl of 5 µM ON-TARGETplus human Rab10 siRNA SMARTpool (GE Dharmacon, Lafayette, CO) was mixed with 320 µl of media and added to the wells. Non-targeting or GAPDH-targeting siRNA (GE Dharmacon) was added to control wells. After 72 h, 200 µl of media containing A. phagocytophilum DC organisms that had been released from infected RF/6A cells was added, and the bacteria were spun on the cells as described earlier. Additionally, cells from one well of each siRNA treatment were harvested for Western blot analysis to confirm knockdown. At 48 h post-infection, cells were harvested for quantitative PCR, processed for microscopy analyses, or the DC-laden media was added to naïve HEK-293T cells to detect the presence of infectious progeny.
Non targeting or gapdh targeting sirna
Manufactured by Horizon Discovery
Non-targeting or GAPDH-targeting siRNA are synthetic double-stranded RNA molecules used for gene silencing. They function by targeting and degrading specific mRNA sequences, thereby reducing the expression of target genes.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using non targeting or gapdh targeting sirna
1
Rab10 Silencing Affects A. phagocytophilum
2
Quantifying Intracellular Pathogen Infection
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4 × 105 HEK-293T cells were seeded onto 12-mm glass coverslips (Electron Microscopy Sciences). After 16 to 20 h, 80 µL of 5 µM ON-TARGETplus human UBC9 siRNA SMARTpool (GE Dharmacon, Lafayette, CO, USA) was mixed with 320 µL of media and added to the wells. Non-targeting or GAPDH targeting siRNA (GE Dharmacon) was added to control wells. After 72 h, 200 µL of media containing A. phagocytophilum organisms that had been naturally released from infected RF/6A cells. Additionally, cells from one well of each siRNA treatment were harvested for Western blot analysis to confirm knockdown. At 48 h post-infection, cells were processed for microscopy analyses.
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