C57BL/6 MEFs were provided by Dr. D. Coverley (University of York, UK) and were cultured in DMEM (high glucose and pyruvate; ThermoFisher) supplemented with 10% FCS, pen-strep and L-glut as RPMI. For transfections, 5x104 cells per well were seeded in 6 well plates and transfected the next day with ON-TARGETplus SMARTpool siRNA (100nM), miRIDIAN miRNA mimics (50nM), or appropriate controls (all Dharmacon, GE Healthcare) using TransIT-siQUEST transfection reagent (Mirus) and Opti-MEM medium (ThermoFisher) for 6 hours before being replaced with complete DMEM. EL4 cells were grown in RPMI supplemented with 10% FCS and were transfected with miRNA mimics using Neon Nucleofection as per manufacturer’s instructions. Non-targeting control (NTC) siRNAs or mimics were used as controls. Cells were harvested 48 hours after transfection.
On targetplus smartpool sirna
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
ON-TARGETplus SMARTpool siRNAs are a collection of four rationally designed small interfering RNAs (siRNAs) that target a specific gene. The siRNAs are designed to maximize gene silencing and minimize off-target effects.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (4 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Dharmafect 4, by GE Healthcare (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Control sirna, by GE Healthcare (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dharmafect 2 transfection reagent, by GE Healthcare (2 mentions)
Miridian mirna mimics, by GE Healthcare (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Transit siquest transfection reagent, by Mirus Bio (1 mentions)
Gene pulser xcell, by Bio-Rad (1 mentions)
On targetplus non targeting pool, by GE Healthcare (1 mentions)
Silencer select pre designed sirna, by Thermo Fisher Scientific (1 mentions)
Nc sirna, by Qiagen (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Nucleofector technology, by Lonza (1 mentions)
Glass bottomed imaging dishes, by MatTek (1 mentions)
Dharmafect 1, by Thermo Fisher Scientific (1 mentions)
22 protocols using on targetplus smartpool sirna
1
Transfection of Mouse Embryonic Fibroblasts
2
Brachyury, MUC1 Knockdown Protocol
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Cells were transfected with ON-TARGETplus SMARTpool siRNA specific for brachyury, MUC1, and a non-targeting control using DharmaFECT two transfection reagent (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. Cells were incubated for at least 48 h in antibiotic-free medium prior to use.
3
Transfection of siRNAs in Cell Lines
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For cancer cell lines, siRNAs were reverse transfected using Lipofectamine 2000. In brief, siRNAs were incubated with Lipofectamine 2000 and serum-free OptiMEM for 15 min at room temperature in the dark. Cells were then trypsinized and resuspended in 1 ml of OptiMEM and added directly into the siRNA/OptiMEM/Lipofectamine solution to give a plating density of 50%, and then they were incubated for 48 h. For NHFs and mouse fibroblasts, siRNAs were transfected using electroporation with a GenePulser Xcell (Bio-Rad Laboratories). Electroporation was performed according to the manufacturer’s instructions. 200 µl PBS containing 107 cells and 5 µM siRNA was electroporated in a 0.2-cm cuvette using a 150 V, 10 ms, and 1 pulse for NHFs and using the preset 3T3 program (160 V and 500 µF) for mouse fibroblasts. Sequences of custom siRNA oligonucleotides used in this study are as follows: control nontargeting siRNA, 5′-UAGCGACUAAACACAUCAA-3′; RAD18, 5′-GAGCAUGGAUUAUCUAUUCAAUU-3′; RNF8, 5′-GAGAAGCUUACAGAUGUUU-3′; RPA32, 5′-GGCTCCAACCAACATTGTT-3′; NBS1, 5′-GUACGUUGUUGGAAGGAAA-3′; Chk1, 5′-GCGUGCCGUAGACUGUCCA-3′; Polκ, 5′-GUAAAGAGGUUAAGGAAA-3′; Polη, 5′-GCAGAAAGGCAGAAAGUUA-3′; Cdc7, 5′-GCUCAGCAGGAAAGGAGUUdTdT-3′; and Cdc6, 5′-ACAAUUAAGUCUCCUAGCA-3′. siCDK1 was the ON-TARGETplus SMARTpool siRNA from GE Healthcare.
4
Knockdown of Bromodomain Proteins in INS-1 Cells
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Dharmacon ON-TARGETplus SMARTpool siRNA (20 nM) with DharmaFECT formulation #1 reagent (GE Life Sciences, Pittsburgh, PA) was used for Brd2, Brd3 and Brd4 knockdown [32 (link)]. INS-1 cells were seeded in a 24 well culture plate at 25,000 cells/well and, after 3 days, were transfected for 24 hrs. RNA was extracted 72 hrs after start of transfection. Knockdown was estimated from Affymetrix Gene Chip analysis.
5
Modulation of Breast Cancer Cells
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All breast cancer cell lines were obtained from the American Type Culture Collection and cultured in DMEM with 10% heat-inactivated fetal bovine serum. Cell lines were engineered to stably express 10- to 15-fold-higher levels of Mena isoforms more than for wild-type cell lines. For siRNA-mediated knockdown experiments, 25 nM SHIP2– or PTP1B–targeted ON-TARGETplus SMARTpool siRNA (Dharmacon, GE Lifesciences, Lafayette, CO) was transfected in serum-free OptiMeM using Dharmafect4 with assays performed 72 h posttransfection. The ON-TARGETplus nontargeted control pool (25 nM) was used in those conditions labeled “scramble.”
6
MYC Gene Silencing in HT-29 Cells
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ON-TARGETplus SMART pool siRNA (GE Healthcare, Little Chalfont, UK) and Silencer Select Pre-designed siRNA (Thermo Fisher Scientific, Waltham, MA, USA) were used for the knockdown of MYC expression. ON-TARGETplus Non-targeting Pool (GE Healthcare) or Silencer Select Pre-designed siRNA (Thermo Fisher Scientific) was used as a control. HT-29 cells were seeded at a density of 8×104 cells/well on a six-well plate for immunoblot analysis and at a density of 3×103 cells/well on a 96-well plate for cell viability assay, and were transfected for 24 h with 20 nM of each siRNA in Opti-MEM (Thermo Fisher Scientific) with lipofectamine RNAiMAX (Thermo Fisher Scientific), in accordance with the manufacturer’s reverse transfection protocol. After 48 h, the cells were used for further experiments.
7
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.
8
Transfection of Dicer1 and STAT3 Knockdowns
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Cells were transfected with ON-TARGETplus SMART pool siRNA against human Dicer 1 (#23405), human STAT3 (#6774) or control siRNA (GE Healthcare Life Sciences, Chalfont St Giles, UK) using Dharmafect 4 according to manufacturer's instructions. Cells were harvested 72 h after transfection. Cells were transfected with pcDNA3 expression vectors for REDD1 and STAT3 respectively using electroporation with nucleofector technology (Lonza, Basel, Switzerland) according to manufacturer`s instructions. Cells were harvested 24 h after transfection.
9
siRNA Knockdown of AP2M1, CAV-1, and FLOT-1
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siRNA sequences: Dharmacon ON-TARGET plus SMARTpool siRNAs were obtained from GE Healthcare targeting AP2M1 (L-008170-00-0005), CAV-1 (L-003467-00-0005) and FLOT-1 (L‑010636-00-0005). Non-targeting siRNA control, GFP (5'-GGCUACGUCCAGGAGCGCAdTdT-3') was synthesised by MWG.
SKBR3 cells (300,000) were seeded in 35 mm glass- bottomed imaging dishes (MatTek) and incubated for 24 hr in complete medium. The transfection mixture was prepared the following day by mixing: transfection reagent 2.4 μL Dharmafect1 (Fisher, UK) with 237.6 μL OptiMEM (Fisher, UK) and incubating at room temperature for 5 min. Meanwhile 12 μL of 5 μM siRNA was mixed with 228 μL OptiMEM. Diluted Dharmafect1 was then mixed with the diluted siRNA and incubated at room temperature for 30 min. The cell medium (in the 6-well plate) was replaced with 1920 μL of compete medium. The transfection mixture (480 μL) was then added to each well, giving a 25 nM final siRNA concentration. The cells were incubated in their transfection medium at 37°C, 5% CO2 for 48 hr before further experimentation.
SKBR3 cells (300,000) were seeded in 35 mm glass- bottomed imaging dishes (MatTek) and incubated for 24 hr in complete medium. The transfection mixture was prepared the following day by mixing: transfection reagent 2.4 μL Dharmafect1 (Fisher, UK) with 237.6 μL OptiMEM (Fisher, UK) and incubating at room temperature for 5 min. Meanwhile 12 μL of 5 μM siRNA was mixed with 228 μL OptiMEM. Diluted Dharmafect1 was then mixed with the diluted siRNA and incubated at room temperature for 30 min. The cell medium (in the 6-well plate) was replaced with 1920 μL of compete medium. The transfection mixture (480 μL) was then added to each well, giving a 25 nM final siRNA concentration. The cells were incubated in their transfection medium at 37°C, 5% CO2 for 48 hr before further experimentation.
10
siRNA Sequences Targeting GTSE1 and Kif4A
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The siRNA sequences 5′-GUACAAAGAAGGUCACUUA-3′ and 5′-CUACUAUCUAGACUCGAAU-3′ targeted GTSE1 and siRNA 5′-GCAAGAUCCUGAAAGAGAU-3′ (Wandke et al., 2012 (link); Barisic et al., 2014 (link)), 5′-CAAUGUGCUCAGACGUAA-3′ (Stumpff et al., 2012 (link)), 5′-CCAAAUCAUUUGCCGAG-3′ (Stumpff et al., 2012 (link)), 5′-AGGCGUACAUUCUCCCUUA-3′ (Voets et al., 2015 (link)), 5′-GAAGAGGCCCACUGAAGUU-3′ (Voets et al., 2015 (link)), 5′-UGAAAGAGAUGUGCGAUGU3′ (Voets et al., 2015 (link)), and 5′-UGACUCGACUGCUUCAAGA-3′ (Voets et al., 2015 (link)) targeting Kif4A were synthesized by Sigma-Aldrich. The siRNA sequence 5′-GAGUAGAACUAGAAUGUGA-3′ targeting Hec1 was synthesized by QIAGEN. ON-TARGET plus SMARTpool siRNAs targeting GTSE1 (5′-ACACGUGGCUGUAGGAUCU-3′, 5′-GAACUGAACCAACAAGGGA-3′, 5′-UAAAUAAUCCGGUUCCCGA-3′, and 5′-GUACAAAGAAGCUCACUUA-3′) and Kif4A (5′-AGGCGUACAUUCUCCCUUA-3′, 5′-GAAGAGGCCCACUGAAGUU-3′, 5′-UGAAAGAGAUGUGCGAUGU-3′, and 5′-UGACUCGACUGCUUCAAGA-3′) were synthesized by GE Healthcare. Nontargeting control siRNA was synthesized by Bioneer.
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