When the cell culture reached 30 ~ 50% confluence, medium was replaced with serum-free DMEM 1 hour before transfection and cells were transfected with X-tremeGENE siRNA reagents (Roche, Switzerland) according to manufacturer’s instructions. miR-26a-5p mimic and inhibitor were purchased from GenePharma (Shanghai, China) .The sequence of miR-26a-5p mimic NC: 5′-UUCUCCGAACGUGUCACGUTT-3′, the sequence of miR-26a-5p inhibitor NC: 5′-CAGUACUUUUGUGUAGUACAA-3′.
Mir 26a 5p mimic
Manufactured by GenePharma
Sourced in China
The MiR-26a-5p mimic is a synthetic double-stranded RNA molecule designed to mimic the mature form of the microRNA miR-26a-5p. MicroRNAs are small non-coding RNA molecules that play a role in regulating gene expression. The MiR-26a-5p mimic can be used in laboratory research applications to investigate the biological functions of this specific microRNA.
Automatically generated - may contain errors
Lab products found in correlation
Mir 26a 5p inhibitor, by GenePharma (4 mentions)
Mimic nc, by GenePharma (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Agomir nc, by GenePharma (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Pmirglo luciferase vector, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Mir nc, by GenePharma (1 mentions)
Inhibitor nc, by GenePharma (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Mir 26a 5p inhibitor, by Thermo Fisher Scientific (1 mentions)
Mir 26a 5p mimic, by Thermo Fisher Scientific (1 mentions)
Foxy5, by GenePharma (1 mentions)
Mmu mir 26a 5p agomir, by GenePharma (1 mentions)
Foxy5, by Selleck Chemicals (1 mentions)
Complete freund adjuvant (cfa), by Merck Group (1 mentions)
Lipofectamine tm 3000 reagent, by GenePharma (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
12 protocols using mir 26a 5p mimic
1
Transfection of miRNA-26a-5p in Cell Culture
2
miR-26a-5p Agomir and Mimic
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hsa-miR-26a-5p agomir, agomir-NC and miR-26a-5p mimic, mimic-NC were all purchased from GenePharma Co. Ltd. (Shanghai, China). The hsa-miR-26a-5p agomir dissolved concentration was 200 nmol/ml in DEPC-treated Water.
3
Interaction between GAS5, miR-26a-5p, and PTEN
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The Bioinformatics online software Starbase (http://starbase.sysu.edu.cn/index.php ) (28 (link)) and TargetScan (http://www.targetscan.org/vert_71/ ) (29 (link)) were utilized to predict the binding sites of lncRNA GAS5 with miR-26a-5p, miR-26a-5p, and PTEN. The complementary sequences of lncRNA GAS5 and miR-26a-5p, and miR-26a-5p and PTEN were amplified and cloned into the luciferase reporter plasmid pmiR-GLO luciferase vector (Promega, Madison, WI, USA) to construct wild-type (WT) plasmids (GAS5-WT AND PTEN-WT) and corresponding mutant (MUT) plasmids (GAS5-MUT and PTEN-MUT). The constructed plasmids were cotransfected with mimic NC and miR-26a-5p mimic (GenePharma) into human degenerative NPCs CPH170. After 48 h, the luciferase activity was detected.
4
Modulating miR-26a-5p to Study RA-FLS
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MiR-26a-5p mimic, inhibitor, and corresponding negative control (NC) were synthesized by GenePharma, China. RA-FLS were cultured in DMEM (Sigma–Aldrich, U.S.A.) with 10% FBS for 24 h. Then RA-FLS were transfected by MiR-26a-5p mimic, inhibitor, and NC (20 μM) by using lipofectamine 2000 reagent (Invitrogen, U.S.A.) at 60–80% of confluence in well plates. Likewise, cells were grown at 37°C in a humidified atmosphere with the presence of 5% CO2. In addition, RA-FLS cells were treated with the PI3K/Akt inhibitor LY294002 (30 μM) (Sigma–Aldrich, U.S.A.) or LY294002 + MiR-26a-5p mimic. Sequences of MiR-26a-5p mimic, mimic NC, miR-26a-5p inhibitor, and inhibitor NC are listed as follow: MiR-26a-5p mimic sense: 5′-UUCAAGUAAUCCAGGAUAGGCU-3′, anti-sense: 5′-CCUAUCCUGGAUUACUUGAAUU-3′. mimic NC: sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, anti-sense: 5′-ACGUGACACGUUCGGAGAATT -3′. MiR-26a-5p inhibitor: 5′-AGCCUAUCCUGGAUUACUUGAA-3′. Inhibitor NC: 5′-CAGUACU UUUGUGUAGUACAA-3′.
5
Degenerative NPC Regulation by GAS5 and miR-26a-5p
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Degenerative NPCs and normal NPCs provided by Procell Life Science and Technology Co., Ltd. (Wuhan, Hubei, China) (CP-H170, CP-H097) were cultured in Dulbecco's modified Eagle's medium-F12 medium containing 20% fetal bovine serum at 37°C with 5% CO2. The cells at passage 3 were taken for subsequent experiments.
The interference and negative control (NC) of GAS5 [small interfering RNA (siRNA) si-GAS5, si-NC], miR-26a-5p mimic, miR-NC and miR-26a-5p inhibitor, and inhibitor-NC were synthesized by Gene Pharma (Shanghai, China). The synthesized vectors were transfected into degenerative NPCs using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). Cells were pre-treated with 25 μm (27 (link)) Akt inhibitor LY294002 (S1737-5mg; Beyotime Biotechnology Co., Ltd., Shanghai, China) for 6 h and transfected with si-GAS5/si-NC. The following experiments were conducted at 48 h post the transfection.
The interference and negative control (NC) of GAS5 [small interfering RNA (siRNA) si-GAS5, si-NC], miR-26a-5p mimic, miR-NC and miR-26a-5p inhibitor, and inhibitor-NC were synthesized by Gene Pharma (Shanghai, China). The synthesized vectors were transfected into degenerative NPCs using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). Cells were pre-treated with 25 μm (27 (link)) Akt inhibitor LY294002 (S1737-5mg; Beyotime Biotechnology Co., Ltd., Shanghai, China) for 6 h and transfected with si-GAS5/si-NC. The following experiments were conducted at 48 h post the transfection.
6
Transfection of HUVECs with miR-26a-5p
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HUVECs were seeded in 6-well plates (5×105 cells per well) for 24 hours, and then the cells were transfected with the negative control (NC), miR-26a-5p mimic, and miR-26a-5p inhibitor (GenePharma, China). Following mixing of Lipofectamine®2000 (ThermoFisher Scientific, USA) with the NC, miR-26a-5p mimic, or miR-26a-5p inhibitor, the mixture was added to the cells and incubated for 24 hours. Then, the cells were collected for the following experiments.
7
miR-26a-5p Transfection in BMSCs
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BMSCs (3 × 105 cells/well) were seeded into 6-well plates,and until then the cells are completely attached and also replaced by a fresh medium for transfection. The miR-26a-5p mimic and NC mimic were synthesized from GenePharma (Shanghai, China). The miRNAs transfection was performed according to the instruction of lipofectamineTM2000 (Invitrogen, Carlsbad, CA). One microliter of the transfection reagent was diluted with 50 µL serum-free medium and incubated at room temperature for 5 min. Diluted miRNA mixed with diluted transfection reagent was incubated at room temperature for 20 min to form the miRNAs-liposome complexes. 100μL of miRNAs-liposome complexes was directed to each well of the cell culture plate, and the plate was then incubated at 37 °C, with 5% CO2 for 24–48 h.
8
Foxy5 and miR-26a-5p Agomir Protocol
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CFA (#F5881) was purchased from Sigma‐Aldrich. Foxy5(#S6961) was purchased from Selleck. Foxy5 was dissolved in DMSO at a concentration of 1 mg/mL. mmu‐miR‐26a‐5p agomir, agomir‐NC and miR‐26a‐5p mimic, and mimic‐NC were all purchased from GenePharma Co. Ltd. (Shanghai, China). The mmu‐miR‐26a‐5p agomir dissolved concentration was 200 nmoL/mL in DEPC‐treated Water.
9
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'
10
Ascl2 3'UTR Luciferase Assay
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Luciferase reporter experiments were performed in the HEK293 or NIH3T3 cells. The wild type and mutant 3′ UTR segment of the Ascl2 gene was amplified by PCR and inserted to replace the original 3' UTR of the LuxA gene in the vector GP-miRGLO (GenePharma). Using Lipofectamine® 2000 Transfection Reagent (ThermoFisher), cells were transfected with 50 ng Ascl2-3'UTR or Ascl2-3'UTR-MUT (mutation) plasmid and 100 nM miR-26a-5p mimics or control miRNA (NC) (GenePharma). Cells were lysed 24 h post-transfection, and luciferase activity was measured by using dual luciferase assays (E1910, Promega, Wisconsin-Madison, USA) following the manufacturer’s protocol.
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