Cells were transfected with NFATc1-siRNA or non-correlated (NC) siRNA (Qiagen, Germantown, Maryland, USA) as previously reported [13 (link)]. In brief, cells (2.5 × 105) were seeded onto 6-well plates in medium without antibiotics; 24 h later, the transfection of siRNAs was carried out with Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific Carlsbad, CA, USA). All transfections were carried out with 20 μM duplex siRNA in medium without FBS or antibiotics. Then, 6 h later, we added RANKL (50 ng/mL) to the medium. One or two days after transfection, cells were recovered to perform further analyses. Experiments were repeated three times.
Nc sirna
Manufactured by Qiagen
Sourced in Germany, United States
NC) siRNA is a laboratory reagent used for gene silencing experiments. It is a synthetic small interfering RNA (siRNA) that does not target any specific gene, serving as a negative control in RNA interference (RNAi) studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Mir 34a, by Qiagen (2 mentions)
Mir 146a, by Qiagen (2 mentions)
Keap1, by Qiagen (1 mentions)
Fasnsirna1, by Qiagen (1 mentions)
Ho 1 sirna, by Qiagen (1 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 kit, by Thermo Fisher Scientific (1 mentions)
Nc sirna, by Sangon (1 mentions)
Mir 34a inhibitor, by Qiagen (1 mentions)
Mir 34a mimic, by Qiagen (1 mentions)
Mirna mimic, by GenePharma (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
On targetplus smartpool sirna, by GE Healthcare (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Mir 181a, by Qiagen (1 mentions)
12 protocols using nc sirna
1
NFATc1 Knockdown in Osteoclastogenesis
2
Keap1 siRNA Knockdown in RAW264.7 Cells
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The RAW264.7 cells (5 × 105 cells/well) were seeded in 6-well plates for 24 h. Briefly, the siRNA pool for Keap1 (Qiagen) and NC-siRNA (Qiagen) were incubated with Lipofectamine RNAiMAX (Promega) in basal media with no serum or antibiotics and allowed to complex for 10 min at room temperature. Then, the complex was added to the cell suspension of each well (final siRNA pool concentration of 10 nM). Finally, cells were incubated for 24 h in a humidified incubator and then used for the analysis.
3
Comprehensive knockdown of key genes
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Cav-1siRNA6 (SI00299614), Cav-1siRNA7 (SI00 299621), Cav-1siRNA8 (SI00299628), FASNsiRNA1 (SI00059752), FASNsiRNA8 (SI00059759), ACACA siRNA1 (SI00013622), ACACAsiRNA2 (SI00013629), and NCsiRNA (1022076) were purchased from Qiagen; ARsiRNA (s1538) was purchased from Life Technologies. Description of the Cav-1, FASN, ACC1 and AR knockdown is presented in the Supplementary Materials and Methods .
4
siRNA-mediated KRAS knockdown
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Specific siRNAs for KRAS and the scrambled negative control (NC) siRNA were purchased from Qiagen (Germany). Target sequences are available on line (Supplementary Table 2 ). The siRNA duplexes were transfected to 1.5×105 cells in 35mm dishes using Lipofectamine RNAiMAX (Invitrogen) at the concentration of 50 nM for 48 hours according to the manufacturer's instructions.
5
HO-1 Silencing and SP/LPS Treatment
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Cells were transfected with HO-1 siRNA, and Non-Correlated (NC) siRNA (Qiagen, Milan, Italy) as previously reported [25 (link)] In brief, cells (2 X 105) were seeded onto 60-mm dishes in medium without antibiotics; 24 h later, the transfection of siRNAs was carried out with Lipofectamine RNAiMAX (Invitrogen). All transfections were carried out with 20 μM duplex siRNA in medium without FBS and antibiotics. After 24 h, cells were split into 6-well plates to perform further analysis. After 24 h (48 h after transfection), cells were untreated or treated for 24 hours with SP and/or LPS and mRNA analysis were performed by RT-qPCR. Experiments were repeated two times.
6
NFATc1 Knockdown in Osteoclastogenesis
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Cells were transfected with NFATc1 siRNA, and Non-Correlated (NC) siRNA (Qiagen, Hilden, Germany) as previously reported [17 (link)]. In brief, cells (2.5 × 105) were seeded onto 6-well plates in DMEM without antibiotics; 24 h later, the transfection of siRNAs was carried out with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). All transfections were carried out with 20 µM duplex siRNA in medium without FBS and antibiotics. After 24 h, cells were split into 6-well plates and incubated with RANKL (50 ng/mL) for 24 and 72 h to perform further analysis. After this time, mRNA analysis was carried out by qPCR, protein analysis was performed by Western blot, and cell staining was performed by immunofluorescence. Experiments were repeated three times.
7
Knockdown of Bcl-2 and FBP1 in H1975 Cells
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For the knock-down of B-cell leukemia/lymphoma 2 (Bcl-2)-antagonist/killer (Bak) or fructose-1,6-bisphosphatase (FBP1) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA (Bak-siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines. NC-siRNA and FBP1-targeting siRNA (FBP1-siRNA) was designed and manufactured by Sangon Biotech Co., Ltd. (Shanghai, China), and the transfection was performed according to the lipofectamine 2000 kit (12,566,014, Thermo Fisher Scientific, Waltham, MA, USA) instructions. The transfection efficiency was ascertained through real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and western blotting (WB). After 48h of transfection with the si-RNAs, the cells were harvested for subsequent experiments.
8
miRNA Regulation of Cell Cycle
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The oligonucleotides including miRNA mimics, inhibitors, and their negative control oligos were purchased from GenePharma (Shanghai, China). Cells were transiently transfected with 50 nM miR-497 mimic, miR-34a mimic, miR-497 inhibitor, miR-34a inhibitor, CCNE1 siRNA (Qiagen, Germany), or siRNA negative control (NC siRNA; Qiagen) using Lipofectamine 2000 (Invitrogen).
9
Transcription Factor Silencing in Cells
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Cells (5.0 × 10 5 ) were plated in 60-mm tissue culture dishes coated with collagen type I (IWAKI). In parallel, cells were transfected with either negative control (NC) siRNA (1027281; Qiagen, Valencia, CA, USA), ON-TARGETplus SMARTpool siRNA targeting ZEB1 (GE Healthcare, Buckinghamshire, England) or Silencer Select siRNA targeting SNAI1 (Ambion #s13185; ThermoFisher Scientific) using Lipofectamine RNAiMAX Reagent and OPTI-MEM I (Thermo Fisher Scientific), according to the manufacturer's recommendations.
10
Regulation of synoviocyte function
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The cells were grown in 6-well dishes at a starting density of 1 × 105 cells/well until a confluence of 85% in DMEM supplemented with 10% FBS; then, the media were replaced with DMEM 0.5% FBS for 6 h before transfection. Afterwards, synoviocytes were transfected with specific inhibitors of miR-34a, miR-146a, and miR-181a (Qiagen, Hilden, Germany), at the concentration of 50 nM, or with their relative negative controls siRNA (NC) (Qiagen, Hilden, Germany), at the concentration of 5 nM, in serum-free medium for a period of 24 h. Supernatants were removed and synoviocytes immediately harvested or incubated with visfatin (5 and 10 μg/mL) or resistin (50 and 100 ng/mL) for additional 24 h.
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