KIF2A gene silencing was studied to exclude the possibility of an off-target effect by KIF2A-siRNA. The two target sequences of the synthetic oligonucleotides for siRNA 5’-GGCAAAGAGAUUGACCUGG-3’(siRNA-1#) and 5’-CCCUCCUUCAAGAGAUAAUTT-3’ (siRNA-2#) (Ambion, Austin, TX, USA) had similar effects (Additional file 2 : Figure S2), hence the first sequence (50 nM) was employed in our experiments. Nonsense-siRNA (Dharmacon, Lafayette, Colorado, USA) and mock treatment were used as negative controls. MDA-MB-231 cells, upon reaching 2 × 105 cells per well in six-well plates, were transfected with KIF2A–siRNA and Nonsense-siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) in Opti-MEM (Invitrogen). Cells in the mock group were only treated with medium. After 48 hours of treatment, the cells were harvested for gene expression (RT-PCR method see above) and protein expression analysis (Western Blotting method see cancer tissue protein analysis).
Nonsense sirna
Manufactured by Horizon Discovery
Sourced in United States
Nonsense-siRNA is a synthetic short interfering RNA (siRNA) designed for use as a negative control in gene silencing experiments. It has no known targets in the human, mouse or rat genome.
Automatically generated - may contain errors
3 protocols using nonsense sirna
1
Investigating KIF2A Gene Silencing
2
Transient siRNA Knockdown of IL-6 in BMDMs
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Il6 Accell siRNA or non-sense siRNA were obtained from Dharmacon. BMDMs were plated in 12 wells and transiently transfected with 2 μg of siRNAs using Lonza Nucleofector reagent according to the Manufacturer’s instruction. 36 h later, cells were harvested. IL-6 expression was determined by flow cytometry.
3
siRNA Knockdown of TRIM and RNF Proteins
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Rnf125 Accell siRNA, Rnf213 Accell siRNA, Trim21 Accell siRNA, Trim31 Accell siRNA, Trim14 Accell siRNA, Trim47 Accell siRNA, or nonsense siRNA was obtained from Dharmacon. BMDMs or HEK293T cells (for Rnf125 siRNA) were plated in 12 wells and were transiently transfected with 2 µg of siRNAs plus 4 µl Lipofectamine 2000 according to the manufacturer’s instruction. 36 h later, cells were harvested. The cell lysates were blotted for specific antibodies against TRIM14, TRIM21, TRIM31, TRIM47, RNF125, and RNF213, respectively.
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