The cells were transfected with 100 nM of the siGENOME SMARTpool reagents (Dharmacon, Lafayette, CA, USA) containing four pooled siRNA duplexes against human FAK, using Lipofectamine 2000 (Invitrogen, CA, USA), according to the manufacturer instructions. The cells were plated on gelatine-coated coverslips 48 h after siRNA treatment in the presence of 5 μM BB94, and incubated at 37°C in the presence of 5% CO2 for a further 24 h. ECM degradation and invadopodia formation were evaluated as described above.
Sigenome smartpool reagent
Manufactured by Horizon Discovery
Sourced in United States
SiGENOME SMARTpool reagents are a collection of siRNA designed to target specific genes. Each SMARTpool consists of a pool of four individual siRNA duplexes that are pre-designed and pre-tested to provide effective gene silencing.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Opti mem medium, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Sicontrol non targeting pool sirna, by Horizon Discovery (2 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Oligofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Mir 338 5p mimics, by Thermo Fisher Scientific (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Anti h3k27me3, by Cell Signaling Technology (1 mentions)
Anti cleaved notch1, by Cell Signaling Technology (1 mentions)
Anti itch, by Cell Signaling Technology (1 mentions)
Anti yy1, by Cell Signaling Technology (1 mentions)
Anti prmt5, by Cell Signaling Technology (1 mentions)
Anti ezh2, by Cell Signaling Technology (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Jmjd3, by Horizon Discovery (1 mentions)
19 protocols using sigenome smartpool reagent
1
Inhibiting FAK Leads to Invadopodia Formation
2
Transient Transfection of Immature DCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient transfection of immature DCs with 100 nM siRNA or 150 nM miRNA mimics were carried out utilizing Oligofectamine reagent (Life Technologies). SHH, non-targeting siRNA and siGLO Lamin A/C were obtained from Dharmacon as siGENOME™ SMARTpool reagents, which contain a pool of four different double-stranded RNA oligonucleotides. MiR-324-5p, miR-338-5p mimics and negative control mimics were purchased from Ambion (Life Technologies). Transfection efficiency was found to be more than 50% in all the experiments as determined by counting the number of siGLO Lamin A/C positive cells in a microscopic field using fluorescent microscope. 48 h post siRNAs transfection or 24 h post miRNAs transfection, the cells were treated or infected as indicated and processed for analysis.
3
Comprehensive Lipid Metabolism Regulatory Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All general chemicals and reagents were procured from Sigma-Aldrich/ Merck Millipore, HiMedia and Promega. Tissue culture plastic ware was purchased from Jet Biofil or Tarsons India Pvt. Ltd. and Corning Inc. siRNAs were obtained from Dharmacon as siGENOME SMART-pool reagents against Yy1, Prmt5, Notch1, Itch. Oleic acid, HRP-tagged anti-β-ACTIN (A3854), 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were procured from Sigma-Aldrich. Anti-cleaved NOTCH1, anti-ITCH, anti-YY1, anti- α-TUBULIN, anti-PRMT5, anti-EZH2, anti-H3K27me3 antibodies were procured from Cell Signaling Technology (USA). Anti-H4R3me2s antibody was sourced from Abcam. Anti-ADRP and anti-CD36 antibodies were procured from Santa Cruz Biotechnology (USA). Anti-LAMINB1 antibody was purchased from IMGENEX. HRP conjugated anti-rabbit IgG/anti-mouse IgG was obtained from Jackson ImmunoResearch (USA). Lipofectamine 3000 was purchased from Thermo Fisher Scientific. BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene lipid stain was from Molecular Probes (Invitrogen/Thermo Fisher Scientific).
4
Utilizing siRNAs and shRNAs to Modulate Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small interfering RNAs (siRNAs) constructs used were obtained as the siGENOME SMARTpool reagents (Dharmacon, Lafayette, CO). The siRNA constructs used were: CREB1 siRNAs were purchased from Genepharma (Shanghai, China). ERK1 siGENOME SMARTpool (M-003592-03-0005), ERK2 siGENOME SMARTpool (M-003555-04-0005), and non-targeting siRNA pool (D-001206-13-20) as control. Transfection of siRNAs was carried out as described previously [65 (link)]. The shRNA targeting Noxa were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Generation of lentiviruses expressing Noxa or control small hairpin RNAs (shRNAs) were as described previously [30 (link)].
5
Reverse Transfection of siRNA in 2D and 3D Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
siGenome SMARTpool reagents (Dharmacon) were reconstituted and diluted to give a final assay concentration of 25 nM in tissue culture treated (2D) or low attachment (Corning, 3D) 96 well plates. These were allowed to complex with Lipofectamine RNAimax (ThermoFisher) as per the manufacturer’s instructions. DLD1 cells were diluted to a density of 3000 cells per well, layered on to the siRNA–lipid mix and incubated to allow reverse transfection to take place. Plates were then topped up with either medium (2D) or agar–medium mix (3D) and cell proliferation quantified using Alamar Blue (10% v/v) after 72h (2D) or 7 days (3D).
6
Macrophage Transfection for Gene Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient transfection of 0.5 × 106 RAW264.7 macrophages with 5 μg of mentioned plasmid constructs was performed using polyethylenimine (PEI, Sigma-Aldrich). In case of experiments involving siRNA, RAW264.7 macrophages were transfected with carefully titrated concentration of 100 nM siRNA. Abl1, Twist1, non-targeting siRNA, and siGLO Lamin A/C were obtained from Dharmacon as siGENOME SMARTpool reagents, which contain a pool of four different double-stranded RNA oligonucleotides. Transfection efficiency was found to be 70% in all the experiments as determined by the number of siGLO Lamin A/C positive cells in a microscopic field using fluorescent microscope. Further, 36 h post-transfection, the cells were treated or infected as indicated and processed for analysis.
7
Mcl-1 Silencing Sensitizes Melanoma Cells to EGb761
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Melanoma cells were seeded at 1×105 cells per well in 24-well plates and allowed to reach 50% confluence on the day of transfection. The small interfering RNA (siRNA) constructs used were obtained as the siGENOMESMARTpool reagents (Dharmacon), the siGENOMESMARTpool Mcl-1 (M-004501-04-0010). The non-targeting siRNA control, SiConTRol- Non-targeting SiRNA pool (D-001206-13-20) was also obtained from Dharmacon. Cells were transfected with 50 to 100 nmol/L siRNA in Opti-MEM medium (Invitrogen) using LipofectAMINE reagent (Invitrogen) according to the manufacturer’s transfection protocol. Twenty-four hours after transfection, the cells were treated with EGb761 at 400 μg/ml for another 48 h before quantitation of apoptotic cells by measurement of percentage of apoptosis in flow cytometry. Efficiency of siRNA was measured by Western blot analysis.
8
Transient Transfection of Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transiently transfection of RAW 264.7 macrophages with 5 μg of dominant negative mutant forms of TLR2, MSI or overexpression constructs of Jmjd3, MINT and MSI was performed using low m.w. polyethylenimine (Sigma-Aldrich). In case of experiments involving siRNA, RAW 264.7 macrophage cells were transfected with 100 nM siRNA. Jmjd3, Acsl1, Adrp, Fat, Psap, Spen, Msi, Notch1, non-targeting siRNA and siGLO Lamin A/C were obtained from Dharmacon as siGENOME SMARTpool reagents, which contain a pool of four different double-stranded RNA oligonucleotides. Transfection efficiency was found to be 70–80% in all the experiments as determined by counting the number of siGLO Lamin A/C positive cells in a microscopic field using fluorescent microscope. Further, 48 h post-transfection (for experiments with RAW 264.7 cells) or 24–36 h post-transfection (for experiments with peritoneal macrophages), the cells were treated or infected as indicated and processed for analysis.
9
RNAi-Mediated Knockdown of Key Autophagy Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNAi-mediated knockdown of gene expression, we utilized Dharmacon siGENOME SMARTpool reagents [Non-Targeting Control Pool #2 (D-001206-14-05), BECN1 (M-010552-01), ATG5 (M-004374-04), and ATG7 (M-020112-01); Thermo Scientific, Waltham, MA]. Transfections were conducted according to manufacturer’s instructions with modifications described previously [26 ]. Importantly, double-transfection was used as described by Smith et al., to maximize efficiency and duration of knockdown [31 (link)]. iOvCa147-E2 cells were seeded to 6-well plates at a density of 350,000/well whereas the density for CaOV3, OVCAR8, SKOV3, and HeyA8 cells were 200,000/well. Cells were counted and seeded for further experimentation 96 h following initial transfection.
10
Silencing Nrf2 and KEAP1 in HepG2 cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
siRNAs against human NFE2L2 (Nrf2) and KEAP1 were acquired from Dharmacon (ThermoFisher Scientific) as siGENOME SMARTpool reagents, as well as in the form of four individual siRNAs. HepG2 cells were transiently transfected with the siRNAs (50nM) using INTERFERin (Polyplus) as described previously (Fredriksson et al. 2011 (link)).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!