miRIDIAN miRNA Human Mimic Library and miRIDIAN miRNA Inhibitor Library (v. 10.1; Dharmacon, Lafayette, CO, USA) were used to transfect KPL4 and SUM190PT cells with 810 miRNA mimics and 816 miRNA inhibitors. Clear polystyrene 384 well microplates (Sigma Aldrich, St. Louis, MO, USA) were pre-printed with the miRNA libraries to achieve a final concentration of 20 nM. SiLentFect™ Lipid Reagent for RNAi (Bio-Rad Laboratories, Hercules, CA, USA) diluted in OptiMEM (Gibco Invitrogen, Carlsbad, CA) was used for transfection. Cells were seeded in the wells (2000 cells/well) and treated with vehicle, 10 µg/mL trastuzumab (Roche Applied Biosciences), 100 nM Lapatinib Ditosylate (GW-572016, Selleckchem) or a combination of trastuzumab and lapatinib. AllStars Hs Cell Death Control siRNA (Qiagen, Chatsworth, CA, USA) was used as a positive control and miRIDIAN microRNA Mimic Negative Control #1, miRIDIAN microRNA Mimic Negative Control #2, miRIDIAN microRNA Hairpin Inhibitor Negative Control #1 (Dharmacon), Negative Control siRNA, miScript Inhibitor Negative Control, AllStars Negative Control siRNA (Qiagen), anti-miR negative control #1, pre-miR negative control #1, and pre-miR negative control #2 (Ambion Inc., Austin, TX, USA) were used as negative controls. The cells were incubated for 72 h at 37 °C, 5% CO2.
Silentfect lipid reagent for rnai
Manufactured by Bio-Rad
Sourced in United States
SiLentFect™ Lipid Reagent for RNAi is a cationic lipid-based transfection reagent designed for the delivery of small interfering RNA (siRNA) and other nucleic acids into a variety of cell types. The reagent forms complexes with negatively charged nucleic acids, facilitating their uptake into cells and enabling efficient gene silencing through the RNA interference (RNAi) pathway.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (2 mentions)
Western blot, by Cell Signaling Technology (2 mentions)
Puromycin, by Cell Signaling Technology (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Pcmv cat t7 sb100, by Addgene (2 mentions)
Anti sox2 d6d9, by Cell Signaling Technology (2 mentions)
Tracrrna atto 550, by Integrated DNA Technologies (2 mentions)
Custom crrnas, by Integrated DNA Technologies (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lapatinib ditosylate, by Selleck Chemicals (1 mentions)
Trastuzumab, by Selleck Chemicals (1 mentions)
Allstars hs cell death control sirna, by Qiagen (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Trastuzumab, by Roche (1 mentions)
8 protocols using silentfect lipid reagent for rnai
1
High-Throughput miRNA Modulation Screening
2
Quantifying p21 Promoter Activity
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HCT116 cells were transiently transfected with siRNA specific for KIF4A and non-specific control siRNA using siLentFect™ Lipid Reagentfor RNAi (Bio-Rad Laboratories, Inc.), p21 promoter plasmid, renilla luciferase plasmid. Forty-eight hours post transfection, we collected the cells and measured the activities of both firefly luciferase and renilla luciferase according to the dual-luciferase reporter assay system (Promega, Madison, WI, USA). The internal standard for transfection efficiency was normalized to renilla luciferase activity. The plasmid of p21-luc (−2400/+11) was a gift from Dr. Baiqu Huang (The Institute of Genetics and Cytology, Northeast Normal University).
3
HER2 Knockdown in SKBR3 Cells
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SKBR3 cells were transfected with 20 nM siRNAs (Qiagen) of Human HER2 siRNA (Hs_ERBB2_14), Human HER2 siRNA (Hs_ERBB2_15), AllStars Negative Control siRNA, using siLentFect™ Lipid Reagent for RNAi (BIO-RAD) when they reached 50% confluence (24 h after plating). Cells were fixed in 4% formaldehyde at room temperature for 10 min 72 h after transfection for immunoFISH.
4
Cell Proliferation Assays for KIF4A
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CCK-8 analysis and colony formation assay were carried out to determine the function of KIF4A on cell proliferation. HCT116 and DLD1 cells were transfected with siRNA of KIF4A or negative control siRNA using siLentFect™ Lipid Reagentfor RNAi (Bio-Rad Laboratories, Inc.), respectively. Forty-eight hours after transfection, for CCK-8 analysis, ~4 × 103 cells were seeded in each well of 96 well plates and CCK-8 solution was added 24, 48, 72, and 96 h after placing. Cells were incubated at 37 °C for 1 h after 10 μl CCK-8 solution was added. The absorbance at 450 nm was measured. For colony formation assay, 7 × 102 cells were cultured in six-well plate at 37 °C for 14 days, visible colonies were washed twice with PBS, fixed, and stained with 4% paraformaldehyde and crystal violet, respectively. The number of colonies was counted visually.
5
CRISPR/Cas9-Mediated SOX2 Knockout in CWR-R1 Cells
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To generate CRISPR/Cas9-mediated SOX2KO cell lines, parental CWR-R1 cells were co-transfected with pT2-EF1a-Cas9-P2A-puro and pCMV(CAT)T7-SB100 (#34879, Addgene; Watertown, MA) using Lipofectamine 2000 (Invitrogen) to introduce Cas9 expression. Stable constitutive Cas9 expression was accomplished by SB100 transposase integration of EF1a-Cas9-P2A-puro. Cas9-expressing cells were selected for and maintained with puromycin (1 mg/mL, Invitrogen) 48 h after transfection. Constitutive Cas9 expression was confirmed by western blot (# 14697, Cell Signaling Technologies) after 1 week of puromycin selection. Two custom crRNAs (Integrated DNA Technologies; Coralville, IA) targeting the N-terminus of SOX2 were selected using CHOPCHOP software (https://chopchop.cbu.uib.no ) (SOX2 crRNA #1: 5’-CGGGCCCGCAGCAAACTTCG-3’, SOX2 crRNA #2: 5’-CGCCCGCATGTACAACATGA-3’) and were individually complexed with tracrRNA-ATTO 550 (#1075927, Integrated DNA Technologies) at a 1:1 ratio immediately before transfection. A final concentration of 10 nM crRNA:tracrRNA duplexes were transfected into CWRR1-Cas9 cells using siLentFect Lipid Reagent for RNAi (#1703360, BioRad) following manufacturer guidelines. Limited dilution was performed to isolate three clonal knockout cell lines, and successful knockout of SOX2 was validated by western blot (anti-SOX2(D6D9), #3579, Cell Signaling Technologies).
6
CRISPR/Cas9-Mediated SOX2 Knockout in CWR-R1 Cells
7
Transient Transfection of HCT116 Cells
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HCT116 cells were transiently transfected with siLMNB2 and nonspecific siCtrl using siLentFect™ Lipid Reagent for RNAi (Bio-Rad Laboratories, Inc.), p21 promoter plasmid, and Renilla luciferase plasmid. Forty-eight hours post transfection, we collected the cells and measured the activities of both firefly luciferase and Renilla luciferase according to the dual-luciferase reporter assay system (Promega, Madison, WI, USA). The internal standard for transfection efficiency was normalized to Renilla luciferase activity. The p21-luc plasmid (−2400/+11) was a gift from Dr. Baiqu Huang (The Institute of Genetics and Cytology, Northeast Normal University).
8
Investigating LMNB2's Role in Cell Proliferation
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CCK-8 assays were carried out to determine the function of LMNB2 in cell proliferation. HCT116 and DLD1 cells were transfected with siRNA targeting LMNB2 or negative control siRNA using siLentFect™ Lipid Reagent for RNAi (Bio-Rad Laboratories, Inc.), respectively. Forty-eight hours after transfection, for CCK-8 analysis, 4000 cells were seeded in each well of 96-well plates, and CCK-8 solution was added 24, 48, 72, and 96 h after transfection. Cells were incubated at 37 °C for 1 h after 10 μl of CCK-8 solution was added. The absorbance at 450 nm was measured. Five millilitres of cell suspension containing 7x10 2 cells was inoculated into a 60 mm dish for continuous culture until visible clones appeared. Then, the cells were xed with methanol and stained with 0.05% crystal violet solution. After two washes with PBS, the plates were photographed using a digital camera. Positive colony formation, de ned as colonies with more than 50 cells, was con rmed by manual counting.
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