The binding site of miR-133a and RAC1 was predicted using bioinformatics software and a website, http://www.targetscan.org/vert_71/ . PC12 cells were lysed with TRIzol, and 5 μL of the cell lysate was mixed with firefly luciferase buffer and 5 μL of the substrate to measure the fluorescence intensity. Then, the luciferase activity of Renilla luciferase was measured by mixing the cell lysate with the Renilla luciferase buffer and 5 μL of enterococcin substrate. The psiCHECK-2 vector was used to analyze the firefly luciferase activity as an internal reference, and the expression of psiCHECK2-RAC1-3′UTR wild type (WT) served as the control. The targeting relationship and binding site between miR-133a and RAC1 were then predicted. The RAC1 3′UTR sequence containing the binding site for miR-133a was synthesized and RAC1 3′UTR WT plasmid (RAC1-WT) and mutant plasmid (RAC1-MUT) were constructed as per the Plasmid Extraction Kit (Promega Corporation, Madison, WI, USA). RAC1-WT and RAC1-MUT plasmids were mixed with mimic NC and miR-133a mimic, respectively, and co-transfected into PC12 cells. Luciferase activity was determined using a luciferase detection kit (BioVision, San Francisco, CA, USA) and a Glomax 20/20 luminometer (Promega).
Luciferase detection kit
Manufactured by Abcam
Sourced in United States
The Luciferase detection kit is a laboratory tool designed to measure the activity of luciferase, a bioluminescent enzyme. The kit provides the necessary reagents and protocols to quantify luciferase expression in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Glomax 20 20 luminometer, by Promega (10 mentions)
Plasmid extraction kit, by Promega (5 mentions)
Glomax20 20 luminometer fluorescence detector, by Promega (4 mentions)
293t cell, by American Type Culture Collection (3 mentions)
Mimics nc, by GenePharma (2 mentions)
Mimic nc, by GenePharma (2 mentions)
Dual luciferase reporter gene system, by Promega (1 mentions)
Vimentin primary antibody, by Merck Group (1 mentions)
Rt pcr kit, by Merck Group (1 mentions)
E cadherin, by Merck Group (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
β catenin, by Merck Group (1 mentions)
Transwell chamber, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Promega (1 mentions)
Mir 27a mimic, by GenePharma (1 mentions)
Mir 92a mimic, by GenePharma (1 mentions)
Lomax20 20 luminometer fluorescence detector, by Promega (1 mentions)
Mir 129 5p mimic, by GenePharma (1 mentions)
Pmir reporter, by Huayueyang (1 mentions)
21 protocols using luciferase detection kit
1
Validation of miR-133a-RAC1 Interaction
2
Luciferase Assay for SLC26A4-AS1 Regulation
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ITPR1‐2Kb enhancer region was cloned to psiCHECK‐2 luciferase reporter gene plasmids. The plasmids were co‐transfected with oe‐negative control (NC), oe‐SLC26A4‐AS1, sh‐NC, and sh‐SLC26A4‐AS1 into TCL‐1 cells. 48 hours after co‐transfection, the cells were lysed with their Renilla luciferase activity and Firefly luciferase activity detected using luciferase detection kit (K801‐200, BioVision) and Dual‐Luciferase Reporter Gene System (Promega, Madison, WI, USA). The ratio of Firefly luciferase unit (FLU) divided by Renilla luciferase unit (RLU) values, reflected the activation of target gene.
3
Molecular Mechanisms of Cervical Cancer
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Normal cervical epithelial cells H8 and cervical cancer cells HeLa were purchased from the Shanghai Cell Bank; the RPMI 1640 culture medium and fetal bovine serum (FBS) were purchased from GIBCO, USA; the primers of miR-NC, miR-375 mimic, si-NC, si-YAP1, miR-375, and U6 were all purchased from Shanghai Gima Co., Ltd.; wild-type (WT) and mutant (MUT) YAP1 plasmids were synthesized by Shanghai Shenggong Biological Company; the RT-PCR kit, YAP1, E-cadherin, β-catenin, vimentin primary antibody and the corresponding secondary antibody, and Transwell chamber were all purchased from Sigma in the United States; RNA extraction kits, LipofectamineTM 2000, MTT, and apoptosis kits were all purchased from Wuhan Boster Biotech; both Trizol reagent and the luciferase detection kit were purchased from BioVision, USA.
4
miR-103a Regulation of BDNF Expression
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Bioinformatics software (http://www.targetscan.org ) was used to predict the targeting relationship between miR-103a and BDNF and the binding sites between miR-103a and BDNF 3′UTR. The sequence of BDNF 3′UTR promoter containing miR-103a binding site was synthesized, and the BDNF 3′UTR wild-type (WT) plasmid was constructed. On the basis of this plasmid, the BDNF 3′UTR mutant (MUT) plasmid was constructed at the mutation binding site. According to the methods of the plasmid extraction kit (Promega, Madison, Wisconsin USA), the cells in the logarithmic growth were inoculated into 96-well plates and transfected with Lipofectamine 2000 at about 70% cell confluence. BDNF-WT and BDNF-MUT were mixed with mimics NC and miR-103a mimics (Shanghai GenePharma Co., Ltd (Shanghai, China)) respectively, and then co-transfected into 293T cells. After 48 h of transfection, the cells were collected and lysed. The luciferase activity was detected by luciferase detection kit (BioVision, San Francisco, CA, USA) and Glomax 20/20 luminometer (Promega, Madison, Wisconsin USA). The experiment was repeated three times.
5
Validating miR-27a Binding to SFRP1 3'UTR
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Bioinformatics software (http://www.targetscan.org ) was used to predict the targeting relationship between miR-27a and SFRP1 and the binding sites between miR-27a and SFRP1 3′UTR. The sequence of SFRP1 3′UTR promoter containing miR-27a binding site was synthesized, and the SFRP1 3′UTR wild-type (WT) plasmid was constructed. On the basis of this plasmid, the SFRP1 3′UTR mutant (MUT) plasmid was constructed at the mutation binding site. According to the methods of the plasmid extraction kit (Promega, Madison, Wisconsin USA), the cells in the logarithmic growth were inoculated into 96-well plates and transfected with Lipofectamine 2000 at the cell density of about 70%. The plasmids of SFRP1-3′UTR-WT and SFRP1-3′UTR-MUT were mixed with mimics NC and miR-27a mimics (Shanghai GenePharma Co., Ltd, Shanghai, China), respectively, and then co-transfected into 293T cells. The cells were collected and lysed after 48 h of transfection. The luciferase activity was detected by luciferase detection kit (BioVision, San Francisco, CA, USA).
6
Luciferase Assay for lncRNA-miRNA Interaction
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lncRNA MSC-AS1 and CDK6 3′UTR sequences consisting of miR-142 binding were synthesized to establish wild-type (WT) plasmids and mutant-type (MUT) plasmids of lncRNA MSC-AS1 and CD6K 3′UTR. After treatment, the WT and MUT plasmids were mixed with plasmids of NC and miR-142 to co-transfect into 293T cells (ATCC, USA). The collected cells were lysed 48 h later. Luciferase activity was detected using a luciferase detection kit (BioVision, San Francisco, CA, USA) and a Glomax20/20 luminometer (MPC12ison, Promega, WI, USA).
7
Validation of miR-92a Binding to TCF21
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Bioinformatics software http://www.targetscan.org was used to predict the targeting relationship between miR-92a and TCF21 and the binding sites of miR-92a to TCF21 3′UTR. TCF21 3′UTR promoter sequence containing miR-92a binding site was synthesized and TCF21 3′UTR wild type plasmid (TCF21-WT) was constructed. On the basis of this plasmid, the mutant type plasmid TCF21 3′UTR (TCF21-MUT) was constructed. The procedure was carried out according to the instructions of plasmid extraction kit (Promega, Madison, Wisconsin, USA). The cells in the logarithmic growth were inoculated into the 96-well plate and transfected with Lipofectamine 2000 when the cell density was about 70%. TCF21-WT and TCF21-MUT were mixed with mimics NC and miR-92a mimics (Shanghai GenePharma Co., Ltd (Shanghai, China)) respectively, and then co-transfected into 293T cells. After 48 h of transfection, luciferase activity was collected and lysed. The luciferase activity was detected by a Glomax20/20 luminometer (Promega, Madison, Wisconsin, USA) with a luciferase detection kit (BioVision, San Francisco, CA, USA). The experiment was repeated three times.
8
Investigating miR-29 Regulation of TUG1 and PTEN
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TUG1 and phosphatase and tensin homolog (PTEN) 3’untranslated region (3’UTR) sequences containing miR-29 binding site were synthesized, respectively, followed by construction of TUG1 and PTEN 3’UTR wild type (WT) and mutation (MUT) plasmids. Then, the constructed plasmids were delivered with NC and miR-29 plasmids into 293T cells (ATCC, Manassas, Virginia, USA). After 48-h transfection, the cells were lysed. Luciferase activity was detected by a luciferase detection kit (BioVision, SanFrancisco, CA, USA) and Glomax20/20 luminometer (Madison, WI, USA).
9
Transcriptional Activity of KLF5
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The transcriptional activity of KLF5 was determined. The reporter gene plasmids with MKK7 or Slug promoter sequence were co-transfected with KLF5 overexpression or empty plasmid into HEK-293 T cells. After 72 h, the cells were collected and the protein was extracted. A luciferase detection kit (K801-200, BioVision, Mountain View, CA, USA), and a lomax20/20 luminometer fluorescence detector (Promega, Madison, WI, USA) were used to detect luciferase activity. The primer sequences of the MKK7 promoter were as follows: F: 5′-TCGAGCTCTAGGTGGCGTCATCCTT-3; R: 5′-GGGCTGATATCCAGGTTGAGGTCGA-3′; the sequences of the Slug promoter were: F: 5′-TGCGTTCCCAAACCTCACGGA-3′; R: 5′-GCCTTCCCCACAGGCTCCCT-3'.
10
Regulation of SOX6 by miR-129-5p
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The targeting relationship between miR-129-5p and SOX6 and the binding site between miR-129-5p and SOX6 3ʹuntranslated region (UTR) were forecasted by bioinformatics software http://www.targetscan.org . The 3ʹUTR fragment of SOX6 gene was amplified by PCR and cloned into pmirGLO vector to construct the recombinant luciferase reporter plasmid of wild type plasmid (SOX6-WT) and mutant type plasmid (SOX6-MUT). SOX6-WT and SOX6-MUT plasmids were extracted according to the steps of the purchased plasmid extraction kit (Promega, Madison, Wisconsin, USA). The logarithmic cells were inoculated into 96-well plates. When the cell confluence was about 70%, Lipofectamine 2000 was utilized for transfection. SOX6-WT and SOX6-MUT were mixed with mimic NC and miR-129-5p mimic (Shanghai GenePharma Co. Ltd., Shanghai, China) respectively, and then co-transfected to neuronal cells. The cells were amassed and lysed after transfected 48 h, and luciferase activity was detected by luciferase detection kit (BioVision, San Francisco, CA, USA) and Glomax 20/20 luminometer (Promega, Madison, Wisconsin, USA).
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