For cell experiments, the plasmid based siRNA targeting for XIST and XIST overexpression vector were purchased from Shanghai Genepharma Co., Ltd. siRNA-XIST: 5′-AATGGAACGGGCTGAGTTTTAG-3′. MiR-93-5p mimics, miR-93-5p inhibitor and miRNA control were purchased from RiboBio Co., Ltd., Guangzhou, China. Briefly, 100 nM of MiR-93-5p mimics or miR-93-5p inhibitor or 1000 ng plasmid were transfected with each 6-well plate for 48 h using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). For animal experiments, cells were stably transfected with XIST based on lentiviral, constructed by Hanyin Biotechnology Co., Ltd., Shanghai, China. After transfection, the DMEM was replaced with DMEM supplemented with puromycin (3 μg/ml) for excluding non-infected cells. Then, the selected cell were used in the subcutaneous xenograft.
Mir 93 5p inhibitor
Manufactured by RiboBio
Sourced in China
The MiR-93-5p inhibitor is a laboratory product designed to inhibit the expression of the miR-93-5p microRNA. It is intended for use in research applications to study the biological functions and regulatory roles of the miR-93-5p microRNA.
Automatically generated - may contain errors
Lab products found in correlation
Mir 93 5p mimic, by RiboBio (4 mentions)
Control mirna, by RiboBio (1 mentions)
Mimics, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Inhibitor nc, by RiboBio (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Mimic nc, by RiboBio (1 mentions)
Plvx ires neo, by Takara Bio (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using mir 93 5p inhibitor
1
XIST-mediated Regulation of miR-93-5p in Cancer
2
Regulating lncRNA H19 and miR-93-5p in hBSMCs
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The expression of lncRNA H19 in hBSMCs was inhibited with transfection of siRNA against H19 (50 nM). Scrambled siRNA was used as the negative control (Qijing, Wuhan, China). SiRNAs were diluted in riboFECTTMCP reagents (Ribobio, Guangzhou, China). Sequences of H19 siRNA were as follows: sense 5’-CCAACAUCAAAGACACCAUTT-3’, antisense 5’-AUGGUGUCUUUGAUGUUGGTT-3’. The expression of miR-93-5p was upregulated by miR-93-5p mimic and downregulated by miR-93-5p inhibitor (Ribobio, Guangzhou, China) respectively. miR-93-5p mimic or inhibitor was transfected into hBSMCs with riboFECTTMCP reagents. For transfection, hBSMCs were inoculated into 24-well plates with a density of 1×104 cells/well and transfection was conducted when cells grew to 50~60% confluence. The transfection complex was added into DMEM/F12 containing 10% FBS with a final volume of 500 μL. Control cells were treated only with same volume of PBS. The medium containing transfection complex was removed after 6 h, and cells were cultured in DMEM/F12 in the presence of IL-13 for an additional 24 − 72 h. The silencing efficiency of siRNA was examined with qRT-PCR.
3
Modulating lncRNA NORAD in HUVECs
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HUVECs were gained from the American Typical Culture Conservatory (ATCC), which were cultured in DMEM maintained in a 37 °C, 5% CO2 incubator. 10% fetal bovine serum and 10% endothelial cell growth supplement were added to the medium. Small interfering (si) RNA (si-NORAD) sequence of lncRNA NORAD gene and its negative control (si-NC), and overexpression plasmid of pcDNA3.1-NORAD were provided by GenePharma Co, while miR-93-5p mimic, mimic-NC, miR-93-5p inhibitor and inhibitor-NC were obtained from the RIBOBIO Co. When cells grew to 80% confluence, the cell transfection was performed using Lipofectamine 3000 (Invitrogen, USA). After 5 h of culture, a new medium was replaced.
4
Regulation of EC Cells by miR-93-5p and TGFβR2
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The EC cells in logarithmic growth phase were collected for transfection with miR-93-5p mimic, miR-93-5p inhibitor and miR-93-5p mimic + oe-TGFβR2 as well as their corresponding controls (obtained from Guangzhou RiboBio Co., Ltd.) using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), respectively. The transfection concentration was 50 nM. Following 6 h of incubation with 5% CO2 at 37°C, the cells were then continuously cultured in fresh medium for 48 h for use in subsequent experiments. The lentiviral vector was used to construct the TGFβR2 overexpression vector: TGFβR2 cDNA with restriction enzyme sites of KpnI and XhoI, as well as a corresponding control sequence (designed by BLOCK-iT™ RNAi Deshgner website) was synthesized, and then ligated into the lentiviral expression vector pLVX-IRES-neo (Clontech Laboratories, Inc.) by T4 ligase. Following 24 h of culture at 37°C, the expression vector was extracted and sequenced. Finally, the packaged vector and viral particle were used to infect EC cells (2×104 cells) cultured in 5 µg/ml Polybrene cultured in a 12-well plate with a multiplicity of infection (MOI) of 50. At 24 h following transfection, cells were harvested for analysis.
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