Human Nrf2-specific siRNA oligo nucleotides (SMARTpool) were purchased from Dharmacon (Lafayette, CO, USA). The following target specific siRNA sequences were used: 5′-UAAAGUGGCUGCUCAGAAU-3′; 5′-GAGUUACAGUGUCUUAAUA-3′; 5′-UGGAGUAAGUCGAGAAGUA-3′; and 5′-CACCUUAUAUCUCGAAGUU-3′. Non-targeting scrambled 20–25 nt siRNA oligonucleotides (Santa Cruz Biotechnology, Inc.) were used as a control. Transient transfections were performed using DharmaFECT 3 transfection reagent (Dharmacon) according to the manufacturer's instructions. Briefly, siRNA/lipid complexes were added to the wells at a final concentration of 100 nM siRNA and 1 μl/well of DharmaFECT 3. Nrf2 gene expression was determined at 48 h after transfection.
Dharmafect 3 transfection reagent
Manufactured by Horizon Discovery
Sourced in Canada, United States, Germany
DharmaFECT 3 is a transfection reagent designed for the delivery of nucleic acids, such as siRNA and plasmid DNA, into mammalian cells. It is a cationic lipid-based formulation that facilitates the uptake of these molecules into the cells.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv6 ac gfp with insert hspa1a, by OriGene (2 mentions)
Pcmv6 ac gfp without insert, by OriGene (2 mentions)
On targetplus smartpool sirna, by Horizon Discovery (2 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (2 mentions)
Mir 140 5p mimic, by Thermo Fisher Scientific (1 mentions)
Multidrop dispenser, by Thermo Fisher Scientific (1 mentions)
On targetplus, by Horizon Discovery (1 mentions)
Sirnas, by GenePharma (1 mentions)
Siglo risc free control sirna, by Horizon Discovery (1 mentions)
Scramble sirna, by Horizon Discovery (1 mentions)
Non targeting control sirna, by Horizon Discovery (1 mentions)
Sirna buffer, by Horizon Discovery (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Axioplan epifluorescent microscope, by Zeiss (1 mentions)
On targetplus sicontrol non targeting pool, by Horizon Discovery (1 mentions)
Mirvana mirna mimic, by Thermo Fisher Scientific (1 mentions)
Antimir29b, by Qiagen (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
18 protocols using dharmafect 3 transfection reagent
1
Silencing Nrf2 Gene Expression
2
miRNA Mimic Transfection in Hypothalamic Cells
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For 24 h miRNA mimic transfection, mHypoE-46 and mHypoE-41 cells were grown to 70–80% confluency in 60 mm tissue culture plates. mirVana miRNA mimic negative control #1 (ID: 4464061), miR-29b-1-5p mimic (ID: MC12431), miR-140-5p mimic (ID: MC10205), miR-143-3p mimic (ID: MC10883), let-7f-1-3p (ID: MC13032), and let-7b-3p (ID: MC12489) were purchased from Thermo Fisher Scientific (Burlington, ON, Canada). For transient transfection, 25 nM mimic or negative control solutions were complexed with Dharmafect 3 Transfection Reagent (Dharmacon, Cedarlane, Burlington, ON, Canada) for 20 min at room temperature in serum- and antibiotic-free plain DMEM with 4500 mg/L glucose (Millipore Sigma). The complexed transfection mixture was then diluted 1:10 with antibiotic-free DMEM with 4500 mg/L glucose (Millipore Sigma) supplemented with 2% FBS. Cells were washed with PBS, and the transfection media was added to the cells for 24 h.
3
High-Throughput siRNA Screening for Adipogenesis
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Confluent hASC were trypsinized and reverse transfection was performed using an automated setup (Multidrop dispenser, Thermo Fisher Scientific, Waltham, MA USA) and BioMek plate aspirator/washer (Beckman Coulter, Indianapolis, IN, USA) in 96-well plates. Dharmafect3 transfection reagent (0.35 μL) (GE Healthcare Dharmacon Inc, Lafayette, CO, USA) was mixed with a specific small interfering RNA (siRNA) pool (ON-Target plus, GE Healthcare Dharmacon Inc), reaching a final concentration in cell culture media of 50nM. After 20 minutes of incubation, 10 000 cells in proliferation medium were added to each well in a 96-well plate. A full list of siRNAs containing the order numbers and sequences of the 148 selected transcriptional regulators, nontargeting siRNA (Negative Control) pool, and siRNA targeting the known adipogenic TFs, such as PPARG and CEBPA, are given in Supplemental Table 1 (38 (link)). Each siRNA pool was run in triplicate. PPARG (n = 3) and nontargeting control (n = 6) were included on each test plate (in total 9 plates were utilized for 1 screen). At 24 hours after transfection, medium was changed to differentiation medium and the cells differentiated until day 9 as previously described (37 (link)). In addition to the 148 siRNAs used in the screen, siRNA targeting glucocorticoid receptor (GR) was used for optimization experiments (Supplemental Table 1) (38 (link)).
4
Silencing Gene Expression in BMDMs
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siRNAs (GenePharma) were mixed with DharmaFECT 3 transfection reagent (Catalog# T-2003-03, Dharmacon) per the manufacturer’s instruction and added to the BMDM at a final concentration of 20 nM71 (link). After incubation with the siRNA complex for 24 h, BMDM were primed with LPS for 20 h and then stimulated with inflammasome inducers as indicated in the figure legends. Details of the sequences of siRNA are listed in Supplementary Table 4 .
5
Knockdown of AMPK, CRBN, and SHP-1 in IgE-sensitized BMMCs
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Genetic knockdown was performed using mouse-specific siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a non-specific siRNA (ThermoFisher, Santa Cruz Biotechnology, Santa Cruz, CA, USA). IgE-sensitized BMMCs were cultured in serum-free medium for 16 h and transfected with 100 nM of AMPKα (50 nM α1(#sc-29674) and 50 nM α2 (#sc-38924)), CRBN (#sc-142562), and SHP-1 (#sc-29479) siRNAs or a non-specific siRNA (Mock) (#12935300), as a negative control using DharmaFECT3 Transfection reagent (Dharmacon, Lafayette, CO, #T-2003-03). After 48 h, cells were stimulated with DNP-HSA for indicated times [32 (link)].
6
Silencing YY1 Transcription Factor
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YY1 silencing was achieved by using Dicer-Substrate siRNA duplexes (IDT). PAC1 cells were transfected with 100 nM YY1 siRNA (siYY1) duplex (5′-AGCAAACUUCUUAUUACAACCGUCGAA-3′) or scrambled siRNA (scr) duplex, the universal negative control, 5′-CGACGGUUGUAAUAAGAAGUUUGCT-3′) at 60% confluence using DharmaFECT3 transfection reagent (Dharmacon). 48 h after transfection, total RNAs were isolated for qPCR assays and total proteins were extracted for Western blotting assays.
7
Efficient siRNA Transfection of C2C12 Myotubes
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C2C12 myotubes were transfected with scramble siRNA (GE Dharmacon, Lafayette, CO, USA) or gp130 siRNA (Santa Cruz Biotech, Santa Cruz, CA, USA) 3 days post-differentiation using DharmaFECT 3 transfection reagent (GE Dharmacon, Lafayette, CO, USA), according to the manufacturer's instructions and as previously described [43 (link)]. Briefly, siRNA and transfection reagent were separately diluted in serum-free and antibiotics-free DMEM and incubated at room temperature for 5 min [43 (link)]. The diluted transfection reagent was then added to the siRNA mixture and allowed to complex with siRNA for 20 min. siRNA-transfection reagent complexes were then added to the antibiotics-free differentiation medium, and myotubes were incubated for 24 hours in a transfection-containing medium (100 nM siRNA concentration). Transfection efficiency was validated by cotransfecting 20 nM (final concentration) of siGLO RISC-Free Control siRNA (GE Dharmacon, Lafayette, CO, USA). Fluorescence was visualized by a Cy3 filter to determine the transfection efficiency. Validated transfected myotubes were collected for protein analysis.
8
Optimized siRNA Knockdown in MDMs
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MDMs were generated as previously described and seeded at 105,000 cells/cm2. Transfection with DharmaFect 3 transfection reagent (Dharmacon) was performed as per manufactures instructions. For each gene a mix of 4 specific “On-target plus Smart-pool” siRNAs was compared with a similar pool of 4 non-targeting control siRNA (Dharmacon). Transfection was performed in antibiotic/serum free RPMI 1640 supplemented with 2mM glutamine in 12 well plates. 5μl/well of DharmaFect 3 was suspended in the appropriate concentration of serum free media. The siRNA was suspended in siRNA buffer (Dharmacon) and diluted in media to a final concentration of 25nM. The transfection reagent and siRNA were combined by gentle agitation at 20°C for 20mins, cells transfected for 8 hours then rested for 12hrs in RPMI 1640 (2mM glutamine, 10μg/ml ampicillin, 10% heat FCS). Transfection efficiency as measured by uptake of florescence (FAM) labelled siRNA was > 95%. The described protocol was optimised for cell seeding density, transfection reagent concentration and transfection duration and yielded target gene expression compared to control of 30% for TDAG-8 and 49% for OGR-1.
9
Knocking down Hsp70 and Hsc70 via RNAi
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To knock down Hsp70 and Hsc70 expression, and prevent induction during treatments, RNA interference experiments were performed using ON-TARGET plus SMARTpool siRNAs (GE Healthcare/Dharmacon, Freiburg, Germany). Cells are cultured in antibiotic-free medium. Then, 24h after seeding, cells were transfected with siRNA and DharmaFECT 3 Transfection Reagent (GE Healthcare/Dharmacon, Freiburg, Germany) according to the manufacturer's instructions. For Hsp70 knockdown 12.5 nM Hspa1a siRNA and 12.5 nM Hspa1b siRNA were used. Hsc70 knockdown was performed using 6.25 nM Hspa8 siRNA. Furthermore, non-targeting scrambled siRNA was used as negative control. Appropriate siRNA concentrations were tested before on cell viability using MTT assay (Fig. 3B ).
To express an Hsp70-GFP fusion protein, the cells were transfected using TurboFect Transfection Reagent (Thermo Fisher Scientific, Schwerte, Germany) 24h after seeding. The vector was purchased from Origene (Rockville, USA): pCMV6-AC-GFP with Insert HSPA1A (human heat shock 70 kDa protein 1A) (RG200270, Origene, Rockville, USA). A pCMV6-AC-GFP without Insert (PS100010, Origene, Rockville, USA) transfected with the same conditions was used as control.
To express an Hsp70-GFP fusion protein, the cells were transfected using TurboFect Transfection Reagent (Thermo Fisher Scientific, Schwerte, Germany) 24h after seeding. The vector was purchased from Origene (Rockville, USA): pCMV6-AC-GFP with Insert HSPA1A (human heat shock 70 kDa protein 1A) (RG200270, Origene, Rockville, USA). A pCMV6-AC-GFP without Insert (PS100010, Origene, Rockville, USA) transfected with the same conditions was used as control.
10
siRNA Knockdown in Preadipocyte Differentiation
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