Cells were transfected to knockdown the expression of ALKBH5, YAP, YTHDF1, YTHDF2, and miR-181b-5p using Lipofectamine TM 3000 Transfection Reagent (Cat# L3000-015; Invitrogen, California, USA) according to the manufacturer’s instructions. Briefly, 2 × 105 cells were seed in a 6-well plate and they will be 70% confluent at the time of transfection. 6 μL Lipofectamine TM 3000 reagent and 40 nM siRNA (Gene Pharma, Shanghai, China) were diluted in 125 μL Opti-MEM (Cat# 31985-070; gibco, Grand Island, USA) medium respectively. After Mix and incubation for 2 min separately, these two regents were then mixed and incubated for another 10 min and then added it to cells. Subsequent experimental measurements were performed 24 h after transfection. The siRNAs and miR-181b-5p inhibitor sequence used are as following:
Lipofectamine tm 3000 reagent
Manufactured by GenePharma
Sourced in China
Lipofectamine TM 3000 reagent is a lipid-based transfection reagent used for the delivery of nucleic acids, such as DNA and RNA, into eukaryotic cells. It facilitates the uptake of genetic material into the cells, enabling researchers to study gene expression and function.
Automatically generated - may contain errors
2 protocols using lipofectamine tm 3000 reagent
1
Knockdown of ALKBH5, YAP, YTHDF1, YTHDF2, and miR-181b-5p
2
Knockdown and Overexpression of METTL1 and miR-26a-5p
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Cells were transfected to knock down the expression of METTL1 and AMO-miR-26a-5p using Lipofectamine TM 3000 Transfection Reagent (Cat# L3000-015; Invitrogen, California, USA). METTL1, FTH1, FTH1-mut plasmid and miR-26a-5p mimic were purchased from Gene Pharma (China) to increase the expression of the plasmid. Brie y, for siRNAs, 2 × 10 5 cells were seeded in a 6-well plate and they will be 70% con uent at the time of transfection 6 µL Lipofectamine TM 3000 reagent and 20 nM siRNA (Gene Pharma, Shanghai, China) were diluted in 125 µL Opti-MEM (Cat# 31985-070; gibco, Grand Island, USA) medium respectively. After Mix and incubating for 2 min separately, these two regents were then mixed and incubated for another 10 min and then added to cells. For plasmids, cells were transfected with 500 ng plasmid using Lipofectamine TM 3000 Transfection Reagent according to the manufacturers' protocols. Subsequent experimental measurements were performed 24 h after transfection.
The sequences used are as following: METTL1 siRNA sense: 5'-CCAAAGGAUAAGAAAGAAATT-3' METTL1 siRNA antisense: 5'-UUUCUUUCUUAUCCUUUGGTT-3' AMO-MiR-26a-5p: 5'-AGCCUAUCCUGGAUUACUUGAA-3' hsa-miR-26a-5p mimics sense: 5'-UUCAAGUAAUCCAGGAUAGGCU-3' hsa-miR-26a-5p mimics antisense: 5'-AGCCUAUCCUGGAUUACUUGAA-3'
The sequences used are as following: METTL1 siRNA sense: 5'-CCAAAGGAUAAGAAAGAAATT-3' METTL1 siRNA antisense: 5'-UUUCUUUCUUAUCCUUUGGTT-3' AMO-MiR-26a-5p: 5'-AGCCUAUCCUGGAUUACUUGAA-3' hsa-miR-26a-5p mimics sense: 5'-UUCAAGUAAUCCAGGAUAGGCU-3' hsa-miR-26a-5p mimics antisense: 5'-AGCCUAUCCUGGAUUACUUGAA-3'
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