The c-Abl short interfering RNAs (siRNAs) were obtained from Invitrogen (Stealth, Carlsbad, CA, USA; cat. no. 1299003), and non-targeting siRNA (Qiagen, cat. no. 1027281) was used as control. Transfection of the cells was performed with 75 nM of three individual siRNAs targeting c-Abl and control siRNA using the siLentFect Lipid Reagent (Bio-Rad).
Non targeting sirna
Manufactured by Qiagen
Sourced in Spain
Non-targeting siRNA is a laboratory tool used for control experiments in RNA interference (RNAi) studies. It serves as a negative control, allowing researchers to differentiate specific target gene knockdown effects from non-specific or off-target effects during RNAi experiments.
Automatically generated - may contain errors
Lab products found in correlation
Nucleofection, by Lonza (2 mentions)
Silentfect lipid reagent, by Bio-Rad (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Interferin, by Polyplus Transfection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dharmafect 1 reagent, by Horizon Discovery (1 mentions)
Sirna universal buffer, by Horizon Discovery (1 mentions)
Sirna transfection reagent, by Qiagen (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Jetoptimus dna transfection reagent, by Polyplus Transfection (1 mentions)
9 protocols using non targeting sirna
1
c-Abl Silencing Using siRNA
2
Knockdown of TSG101 and STAM1 in T cells and DCs
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Small interference RNA-mediated knockdown of TSG101 or STAM1 was performed using siRNA-TSG101 or siRNA-STAM1 (Santa Cruz Biotechnology). A non-targeting siRNA (Qiagen, Valencia, CA) was used as the negative control. siRNAs were transfected into T cells and DCs by nucleofection (Lonza Wakersville Inc, Wakersville, MD) per manufacturer’s instructions.
3
siRNA Transfection in Hypoxic eAC Cultures
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Design of siRNA (Supplemental Table S1 ) was performed by Eurogentec service, and matching was confirmed using NCBI’s Basic Local Alignment Search Tool (BLAST). Sequences and BLAST results are presented in Figure 5 A,B and represented with CLC Sequence Viewer 7.7 software (Qiagen, Manchester, UK). Mammalian siRNA duplexes’ negative control was used as non-targeting siRNA (Qiagen). eAC were seeded into type I/III collagen scaffolds, as described earlier. At D0, eAC were transfected with a mix (150 μL/scaffolds) of INTERFERin (Polyplus-transfection), OptiMEM (Thermofisher), and small INTERFERing RNA (siRNA) for 10 min at room temperature, according to the manufacturer’s instructions. Quantities of 2, 5, 10, or 25 nM of siRNA were transfected. Then, the INTERFERin siRNA complex was slowly added to scaffolds disposed in 450 μL of 3D medium, pre-equilibrated to 3% O2 by bubbling, with or without 50 ng/mL of BMP-2. eAC were then cultured in hypoxia (2–4% O2) for 7 days. The first medium change for siRNA medium elimination was performed two days after transfection.
4
Osteoclast Differentiation via siRNA
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BMMs were seeded into 48-well plates at a density of 3.5 × 104 cells/well with 30 ng/ml M-CSF. After 24 h, cells were transfected with 40 nM mouse JNK on-target plus smart pool siRNAs (5′-AAAGAATGTCCTACCTTCT-3′) (QIAGEN, Hilden, Germany) or mouse JAK3 on-target plus smart pool siRNAs (5′-CACGTTAGACTTTGCCATCCA-3′; QIAGEN) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The control contained 40 nM non-targeting siRNA (QIAGEN). The transfection took place in 2.5 ml of serum-free medium for 6 h; the cells were then cultured for 4 days in complete media containing 30 ng/ml M-CSF and 100 ng/ml RANKL for osteoclast formation.
5
LARG Knockdown in iMDDCs
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Small interference RNA-mediated knockdown of LARG was performed using LARG –siRNA (Santa Cruz Biotechnology). A non-targeting siRNA (Qiagen, Valencia, CA) was used as the negative control. siRNAs were transfected into iMDDCs by nucleofection (Lonza Wakersville Inc, Wakersville, MD) per manufacturer’s instructions.
6
Gene Silencing with siRNA Duplexes
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Grb2 (Jiang et al., 2003 (link)), c-Cbl and Cbl-b (Huang et al., 2006 (link)) siRNA duplexes were resuspended in 1 × siRNA universal buffer (Dharmacon, Lafayette, Colorado) to 20 μM before transfection. Nontargeting siRNA was from Qiagen. Parental and derived cell lines grown in 60-mm dishes (60–80% confluency) were transfected with siRNA duplexes at a final concentration of 100 nM with DharmaFECT I reagent (Dharmacon) following manufacturer’s protocol. After 24 h, the cells were replated to 100-mm dishes and used for experiments 2 d after.
7
Comprehensive siRNA Targeting of Endocytic Proteins
8
Investigating GPR30 Knockdown in GC-2 Cells
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GPR30 siRNA and non targeting siRNA were purchased from Qiagen. GC-2 cells were plated into 60 mm dishes at 1 × 106 cells for protein extraction, and into 24-well plates at 2 × 105 cells/well for proliferation assay and used for transfection 24 h later. The cells were transfected using the siRNA transfection reagent (Qiagen, Biotechnology) with 10 nM GPR30 or control siRNA according to the manufacturer's instructions. The oligonucleotides used were: 5′-TGGAGTAGTCGCATCCAT-3′ for GPR30 and 5′-GATCTCAGC ACGGCAAAT-3′ for the scrambled control. Immunoblotting and the quantitative real-time reverse transcription-polymerase chain reaction (qPCR) were then performed. To confirm the specific inhibitory activity, we carried out a Western blot analysis using the antibody against GPR30. For inhibited proliferation experiments, cells were maintained in medium containing the transfection mix for 24 h and medium was then replaced for the treatment. Proliferation was evaluated 48 h later.
9
siRNA and Plasmid Transfection Protocols
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For siRNA, RNAimax lipofectamine (Invitrogen, Waltham, MA) was used to transfect HNRNPK smartpool siRNA (Dharmacon) or non-targeting siRNA (Qiagen) according to the manufacturer's protocol. For overexpression plasmids, jetOptimus DNA transfection reagent (Polyplus, Illkirch, France) was used according to the manufacturer's protocol.
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