Cells were transfected with the indicated miRNA mimics or inhibitors (50 nM) or control oligonucleotides (50 nM) (Thermo Fisher Scientific) using the Oligofectamine reagent (Invitrogen). At 48 hours after transfection, cells were harvested for RNA and protein analyses.
Control oligonucleotides
Manufactured by Thermo Fisher Scientific
Control oligonucleotides are short, synthetic DNA or RNA sequences used to validate the performance of laboratory assays and instruments. They serve as positive or negative controls to ensure the reliability and accuracy of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Oligofectamine reagent, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Sirna oligonucleotide, by Thermo Fisher Scientific (1 mentions)
Stealth sirna oligonucleotides, by Thermo Fisher Scientific (1 mentions)
5 protocols using control oligonucleotides
1
miRNA Transfection and Expression Analysis
2
Antibody Characterization and miRNA Assays
3
Knockdown and Overexpression of GPx7 in Cells
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Cells were plated on 60-mm diameter dishes 24 h before transfection. The following double-stranded stealth siRNA oligonucleotides (Snata Cruz Biotechnologh) were used: mouse GPx7 siRNA oligonucleotides (sc-78832). Control oligonucleotides with comparable GC content were also obtained from Invitrogen. For knockdown, cells were transfected with control or gene-specific siRNA at 60 nM in OPTI-MEM medium using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s protocol. The next day, the medium was replaced with fresh DMEM containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, and the cells were incubated for 24 h before harvest. Total RNA extracts were prepared from the cell at the indicated time point (2 days), and RT-PCR or real-time PCR was performed. For the overexpression, the cells were plated at a density of 5 × 105 cells/well. The plasmid, GPx7 overexpressing vector (pcDNA3-GPx7-FLAG) was packaged with Lipofectamine 2000 (Invitrogen), and cells were transfected with either a control or GPx7-overexpressing vector according to the manufacturer’s instructions.
4
SCARA5 RNAi Knockdown in A33 Cells
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The A33 cells were plated into 60-mm-diameter dishes 48 h before transfection. The following double-stranded stealth siRNA oligonucleotides (Invitrogen, Carlsbad, CA, USA) were used: mouse SCARA5, sense 5′-GGGAC CGAAC AGGAC AGCAG AGUGA-3′ and antisense 5′-UCACU CUGCU GUCCU GUUCG GUCCC-3′, a set of three validated siRNA oligonucleotides. Control oligonucleotides with comparable GC content were also from Invitrogen. For knockdown, cells were transfected with control or gene-specific siRNA at 20 nM in OPTI-MEM medium using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s protocol. The next day, the medium was replaced with fresh DMEM containing 10% calf serum, and the cells were incubated for 48 h before the induction of differentiation. Oil red-O staining of SCARA5 knockdown was performed at day 5.
5
HIF1α Knockdown in Hypoxia
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Cells were seeded at 20–40% confluency and the day after transfected with 25 nM siRNA using Lipofectamine RNAiMAX according to the manufacturer’s protocol. One day later, cells were incubated under normoxia or hypoxia with fresh McCoy's 5A Medium containing 10% FBS and processed for designated assays. The following stealth siRNA oligonucleotides (Invitrogen) were used for human HIF1α knockdown: 5‘- GGGAUUAACUCAGUUUGAACUAACU-3′ (sense) and 5′- AGUUAGUUCAAACUGAGUUAAUCCC-3’. Control oligonucleotides with comparable GC content were also purchased from Invitrogen.
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