For transfection, miR-34a mimics, miR-34a inhibitor, and negative controls (Genepharma Co. Shanghai) were transfected using lipofectamine 2000 (Invitrogen, US) according to the manufacturer's protocol. The sequences of miR-34a mimics, miR-34a inhibitor, and controls were listed in Supplementary Table 1 . We measured the proliferation activity of HIBEC by using cell counting kit-8 (CCK8, Biosharp Co., Guangzhou). 10 μl CCK8 was added to each well. The absorbance was measured. The results were shown as optical density measured at 450 nm.
Mir 34a inhibitor
Manufactured by GenePharma
Sourced in China
The MiR-34a inhibitor is a laboratory product that functions to inhibit the activity of the microRNA-34a (miR-34a) molecule. miR-34a is a small non-coding RNA that plays a role in the regulation of gene expression. The MiR-34a inhibitor can be used in research applications to study the biological functions and mechanisms of miR-34a.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Mir 34a mimic, by GenePharma (5 mentions)
Cck 8, by Biosharp (1 mentions)
X treme sirna transfection reagent, by Roche (1 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Sw480, by National Collection of Authenticated Cell Cultures (1 mentions)
Sw620, by National Collection of Authenticated Cell Cultures (1 mentions)
Hct116, by National Collection of Authenticated Cell Cultures (1 mentions)
Oxamate, by Merck Group (1 mentions)
Mouse monoclonal antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Mir control, by GenePharma (1 mentions)
Synthetic mir 34a mimic, by GenePharma (1 mentions)
Hotair sirna, by GenePharma (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Pcdna3.0 vector, by Thermo Fisher Scientific (1 mentions)
Mir 34a mimic, by Thermo Fisher Scientific (1 mentions)
8 protocols using mir 34a inhibitor
1
Transfection and Proliferation Assay
2
Transfection of MSCs with miR-34a and SIRT1 siRNA
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Before transfection, MSCs were replayed into six-well plates at a density of 2 × 105 cells/well and incubated overnight. For over-expression or inhibition of miR-34a, cells were transfected with 20 nM of miR-34a mimic or miR-34a inhibitor (both from GenePharma Co., Ltd., Shanghai, China), respectively. For SIRT1 inhibition, 100 nM Akt siRNA (GenePharma Co., Ltd., Shanghai, China) was transfected into cells. All miRNAs and siRNA were transfected into MSCs using a commercial transfection reagent (X-treme siRNA transfection reagent; Roche Applied Science, Penzberg, Germany) according to the manufacturer’s protocol. Forty-eight or 72 h after transfection, the cells were harvested for further analysis.
3
Modulating Autophagy in Colorectal Cancer
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The human colon epithelial cell line FHC obtained from the American Type Culture Collection (ATCC) was cultured in DMEM/F12 medium (GIBCO; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO2 atmosphere. The human CRC cell lines HCT116, HT29, SW620 and SW480 were purchased from the Cell Bank of the Chinese Academy of Sciences and were routinely cultured using RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS. The miR-34a mimics or miR-34a inhibitor purchased from Shanghai GenePharma Co., Ltd., were transfected into the CRC cells for 48 h using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) in indicated concentrations according to the supplier's instructions. Rapamycin (10 nM; Cell Signaling Technology, Inc.) and 3-methyladenine (3-MA; 5 mM; Sigma-Aldrich; Merck KGaA) were added to further activate or inhibit the flux of autophagy in CRC cells as previously described (12 (link)).
4
Examining LDHA and Hexokinase 2 in miR-34a Modulation
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Rabbit monoclonal lactate dehydrogenase-A (LDHA) antibody was ordered from Cell Signaling (#2012); mouse monoclonal antibody against Hexokinase 2 was purchased from Santa Cruz (sc-6521); mouse monoclonal antibody against β-actin was purchased from Santa Cruz Biotechnology (sc-47778). Oxamate was purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). MiR-34a mimic, miR-34a inhibitor, and their corresponding negative control were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China).
5
miRNA Regulation of Colorectal Cancer Cells
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HCT8 and SW480 cells (1 × 105 cells) were seeded in 6‐well plates in DMEM medium for 12 hours. Then, cells were transfected with 10 nmol/L mimic nontargeting control and the relevant miRNA mimic, and 10 nmol/L inhibitor nontargeting control and the relevant miRNA inhibitor through Oligofectamine transfection reagent. Next, HCT8 and SW480 cells (1 × 105 cells) were plated in 6‐well plates and transfected with 5 nmol/L NEAT1 shRNA or shRNA negative control via 0.2% Lipofectamine 3000 in DMEM medium. The synthetic miR‐34a mimic, miR‐34a inhibitor and their negative control (miR‐control), specific shRNA targeting NEAT1 or scramble shRNA, and human HMGB1 overexpressing plasmid were obtained from GenePharma. After 5 hours, the transfection reagent was replaced with DMEM medium. Forty‐eight hours later, cells were collected for further experiments.
6
siRNA and miRNA Inhibitor Transfection
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HOTAIR siRNA, miR-34a inhibitor or their corresponding negative controls (NCs) were obtained from GenePharma Co., Ltd. (Shanghai, China) and transfected into the cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.).
7
Neuroprotection via miR-34a Modulation
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The cells were first transfected with negative control miRNA, miR-34a mimic or miR-34a inhibitor (GenePharma, Shanghai, China) for 24 h using Lipofectamine RNAiMAX (Invitrogen, CA, USA) according to the manufacturer’s introductions. The cells were then treated with TDB (25 μM) under OGD condition for 4 h, whereupon OGD was terminated and the cells were further cultured for 24 h under normoxic conditions. The cells were then assayed by MTT assay, MMP assay and western blot.
8
Regulation of c-Met by miR-34a in Liver Cancer
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Huh7 and HCCLM3 cells (1.0×105 per well) were seeded and grown overnight in six-well plates into 6-well plates (2,000,000 cells/well) in DMEM supplemented with 10% FBS for 24 h at 37°C. miR-34a mimics, miR-34a inhibitor and the corresponding negative controls (NCs) were purchased from Shanghai GenePharma Co., Ltd. In addition, the coding domain sequences of c-Met mRNA were amplified by PCR, and inserted into pcDNA 3.0 vector to enhance its expression (Invitrogen; Thermo Fisher Scientific, Inc.), named as pcDNA-c-Met. pcDNA 3.0 empty vector was used as the control. A total of 100 nM miR-34a mimics, 100 nM miR-34a inhibitor or 2 μg pcDNA-c-Met were transfected into Huh7 and HCCLM3 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following manufacturer's instructions. After 48 h transfection, cells were harvested for subsequent analysis.
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