BHK-21 cells (3 × 104 cells/well) were seeded in 12-well culture plates and incubated overnight. The cells were transfected with the miR-125b-5p mimic (Exiqon, Germantown, MD, USA) at final concentrations of 0.2, 0.5, 1, 5, and 25 nM, with 1 μL of Lipofectamine 2000 reagent (Invitrogen) for each well in Opti-MEM, according to the manufacturer’s protocol. Cell viability was determined at 48 h post-transfection by the conversion of thiazolyl blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to blue formazan crystals. An MTT reagent (500 μL; 0.5 mg/mL, Life Technologies, Carlsbad, CA, USA) was added into each well and incubated at 37 °C for 3 hrs. The reagent was removed, formazan crystals were dissolved in 500 μL dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), and the optical density at 570 nm of 100 μL aliquots was measured by Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific). Values were expressed as a percentage relative to those obtained in scrambled siRNA control. Assays were performed in triplicate.
Mir 125b 5p mimic
Manufactured by Qiagen
Sourced in United States
The MiR-125b-5p mimic is a laboratory reagent designed to mimic the function of the microRNA (miRNA) miR-125b-5p. MiRNAs are small, non-coding RNA molecules that play a role in the regulation of gene expression. The MiR-125b-5p mimic can be used in research applications to study the biological functions and effects of this specific miRNA.
Automatically generated - may contain errors
Lab products found in correlation
Hepatocyte culture medium, by Lonza (2 mentions)
Scramble control, by Qiagen (2 mentions)
Mir 125b 5p inhibitor, by Qiagen (2 mentions)
Liberase, by Roche (2 mentions)
Mtt reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Recombinant epidermal growth factor, by Lonza (1 mentions)
4 protocols using mir 125b 5p mimic
1
miR-125b-5p mimic transfection and cell viability
2
Luciferase Reporting Assay for miRNA Targets
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For luciferase reporter assay, BHK-21 cells (1 × 105 cells/well) were cultured in 12-well plates and transfected with 100 ng/well of the indicated reporter constructs (pmirGLO vector alone, pmirGLO-containing target genes, or pmirGLO-containing mismatch of target genes) in conjunction with a 0.5 nM miR-125b-5p mimic (Exiqon) or 0.5 nM scrambled miRNA (Exiqon) as a negative control using 2 μL of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. At 24 h post-transfection, the cells were washed with PBS and lysed with 250 μL of a Passive Lysis Buffer (Promega), and cell extracts were subjected to the luciferase assay using a dual-luciferase assay system (Promega). After brief centrifugation (10,000× g for 30 s), 20 μL of supernatant were transferred into a tube containing 100 μL Luciferase Assay Reagent II (LAR II) and mixed by pipetting. Firefly luciferase (FLuc) activities were determined in a 20/20 luminometer (Turner Biosystems). Subsequently, 100 μL of Stop & Glo Reagent were added to determine Renilla luciferase activity. Relative luciferase activity was calculated by normalizing the ratio of Firefly/Renilla luciferase.
3
Isolation and Transfection of Primary Hepatocytes
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Freshly isolated primary human hepatocytes were purchased from Cytonet GmBH. Primary hepatocytes from mouse liver were isolated as described26 (link). Briefly, mice were anaesthetized and perfused with Liberase (Roche). After perfusion, livers were disintegrated mechanically before collecting hepatocytes by low-speed centrifugation. Non-parenchymal cells were removed by discarding the supernatant. For all in vitro transfection experiments, we used Percoll density gradient-purified mouse hepatocytes to achieve high transfection efficiency. Ten thousand primary hepatocytes per well of a collagen-coated 12-well plate (BD) were seeded. Twelve hours after seeding, hepatocytes were transfected with 25 nM miR-125b-5p mimic, miR-125b-5p inhibitor or control scramble (Qiagen), using the Targefect reagent in the presence of virofect enhancer (Targeting Systems). Transfected hepatocytes were cultured in Hepatocyte Culture Medium (Lonza).
4
Transfection of Primary Hepatocytes
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Freshly isolated primary human hepatocytes were provided by Regenerative Medicine and Experimental Surgery, Hannover Medical School, Hannover, as reported.(12 ) Mouse primary hepatocytes were isolated from mouse liver as described.(8 ) Briefly, mice were anesthetized, and their livers were perfused with Liberase (Roche). After perfusion, livers were disintegrated mechanically before collecting hepatocytes by low‐speed centrifugation. Nonparenchymal cells were removed by discarding the supernatant. For all in vitro transfection experiments, we used Percoll density gradient‐purified mouse hepatocytes to achieve high transfection efficiency. We seeded 100,000 primary hepatocytes per well of a collagen‐coated 12‐well plate (TPP). Twelve hours after seeding, hepatocytes were transfected with 25 nM or 50 nM miR‐125b‐5p mimic, miR‐125b‐5p inhibitor, control scramble (Qiagen), or 100 nM ABTB1 small interfering RNA (siRNA) (Qiagen) using Targefect reagent in the presence of virofect enhancer (Targeting Systems). Transfected hepatocytes were cultured in hepatocyte culture medium (Lonza) containing recombinant epidermal growth factor (an inducer of hepatocyte proliferation), transferrin, ascorbic acid, insulin, hydrocortisone, bovine serum albumin, and gentamicin sulfate‐amphotericin.
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