RNAi experiments in worms were performed on NGM plates seeded with E. coli HT115 (DE3) bacteria transformed with the indicated RNAi construct (primers listed in Supplementary Table S2 ) or the appropriate empty vector (plasmid L4440 or T444T purchased from Addgene, Watertown, MA, USA). All RNAi assays were performed at 20 °C, as previously described [48 (link)]. For human LonP1 small interfering RNA (siRNA) experiments, dicer-substrate RNA (DsiRNA) duplexes (5′-AAUCAGAGUGUGGCAUAGAAGCUAT-3′) were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA). A predesigned DsiRNA to be used as a negative control was also purchased from IDT. SiRNA transfections were performed using Lipofectamine2000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, 2 × 105 cells (WM266-4 or HT1080) were seeded in a 35 mm culture dish. The next day, 20 nM DsiRNA were transfected with 5 μL Lipofectamine2000 in Opti-MEM (Gibco, Waltham, MA, USA). After 48 h, cells were subcultured (ratio 1:6) and retransfected to a final incubation period of 144 h.
Dsirna duplexes
Manufactured by Integrated DNA Technologies
Sourced in United States
DsiRNA duplexes are double-stranded RNA molecules designed for gene silencing applications. They consist of a guide strand and a passenger strand, each approximately 27 nucleotides in length. DsiRNA duplexes are used to induce RNA interference (RNAi) and downregulate the expression of target genes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Transfectagro, by Integrated DNA Technologies (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Transfectagro, by Thermo Fisher Scientific (2 mentions)
Transfectagro, by Corning (2 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
4 protocols using dsirna duplexes
1
RNAi and siRNA Experiments in Worms and Cells
2
Plasmid and siRNA Transfections in Cell Culture
3
Plasmid and siRNA Transfections in Cell Culture
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Plasmid transfections were performed using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s protocol but using Transfectagro (Corning) instead of Opti-MEM. Cells were incubated with transfection mix in Transfectagro supplemented with 10 % FBS for 6–8 h, following by a change of media to regular growth media and analysis after 18–20 h.
DsiRNA duplexes were obtained from Integrated DNA Technologies. Transfections with siRNA were performed with the appropriate duplexes (seeKey Resources Table ) using Lipofectamine RNAiMAX (Thermo Fisher) following the manufacturer’s protocol except using Transfectagro in place of Opti-MEM. Cells were incubated wit transfection mix in Transfectagro supplemented with 10% FBS for 12–16 h, followed by exchange with fresh media. NC1 (negative control 1, IDT) was used as the control siRNA duplex for all experiments. Forty-eight h post transfection, cells were subjected to analysis via western blot, microscopy or flow cytometry.
DsiRNA duplexes were obtained from Integrated DNA Technologies. Transfections with siRNA were performed with the appropriate duplexes (see
4
siRNA Transfection for Protein Analysis
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DsiRNA duplexes were obtained from Integrated DNA Technologies. Transfections with siRNA were performed using Lipofectamine RNAiMAX with the appropriate duplexes (see Table 1) as described previously (47) (link), and 48 h post transfection, cells were analyzed via Western blot, flow cytometry or other readouts.
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