The wild type (WT, with miR-144-5p binding sites) and mutant-type (MUT, with mutated miR-144-5p binding sites) circ_0066881 and RORA 3’UTR sequences were cloned into the pmirGLO plasmid (Promega, Madison, WI, USA). Novel reporters (circ_0066881 WT, circ_0066881 MUT, RORA 3’UTR WT and RORA 3’UTR MUT) were co-transfected with miR-144-5p or miR-NC for 48 h, then PDLCs were collected for luciferase activity analysis through the Dual-Luciferase Reporter Detection Kit (Promega).
Dual luciferase reporter detection kit
Manufactured by Promega
Sourced in United States, China
The Dual-Luciferase Reporter Detection Kit is a laboratory instrument designed to quantify and analyze the activity of two different luciferase reporter enzymes within a single sample. It provides a rapid and sensitive method for measuring gene expression and transcriptional regulation.
Automatically generated - may contain errors
Lab products found in correlation
Pmirglo plasmid, by Promega (5 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Pmirglo luciferase plasmid, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
293 t cells, by BioVector (1 mentions)
Pmirglo, by Promega (1 mentions)
Rip lysis buffer, by Merck Group (1 mentions)
11 protocols using dual luciferase reporter detection kit
1
miR-144-5p Regulation of circ_0066881 and RORA
2
Dual-Luciferase Assay Validates circRNA-miRNA Interactions
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Interaction analysis was performed using dual-luciferase reporter assay [21 (link)]. The wild-type (WT) sequences of circ_0005526 and TCF4 3ʹUTR were inserted into pmirGLO luciferase plasmid (Promega, Madison, WI, USA), respectively. The constructed plasmids containing miR-142-5p binding sites were named as circ_0005526-WT and TCF4 3ʹUTR-WT. Then miR-142-5p binding sites in circ_0005526 and TCF4 sequences were mutated, and mutant-type (MUT) plasmids (circ_0005526-MUT and TCF4 3ʹUTR-MUT) were used as negative controls. After co-transfection with each plasmid and miR-142-5p or miR-NC for 48 h, luciferase intensity was analyzed through Dual-Luciferase Reporter Detection Kit (E1910; Promega).
3
Evaluating miR-103a-3p Binding in TRIM35
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The TRIM35 3′ UTR containing miR-103a-3p wild-type and mutant binding sites were respectively constructed on the pmirGLO vector. The BEL-7402 and SK-HEP1 cells were cultured until a convergence degree of 70%–90% was achieved, and the miR-103a-3p mimic was co-transfected into the BEL-7402 and SK-HEP1 cells with the vector. After 48 h of culture, the dual luciferase activity was analysed using the dual luciferase reporter detection kit (Promega.USA).
4
circ0006168-mut Dual-Luciferase Assay
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Circular RNA LPAR3‐wt or circ0006168‐mut was constructed into the dual‐luciferase reporter vector (GP‐miRGLO). Kyse450 cells were cultured for 24 hours in a 24‐well plate at the density of 5 × 104 cells/well for transfection. Lipofectamine 2000 (Invitrogen) was used to cotransfect the dual‐luciferase reporter gene plasmid and miR‐198 mimics or miR‐198 mimic‐NC into Kyse450 cells. Forty‐eight hours later, the luciferase activities were measured using the dual‐luciferase reporter detection kit (Promega).
5
Validating circSATB2-miR-330-5p Interaction
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The target prediction was carried out using starBase (http://starbase.sysu.edu.cn ). The circSATB2 sequence (containing the miR‐330‐5p binding sites) was cloned into the pmirGLO plasmid (Promega) to construct the wild‐type (wt) plasmid circSATB2 wt. The mutant sequence of circSATB2 (containing the mutated sites of miR‐330‐5p) was inserted into the luciferase plasmid, which was considered as the mutant‐type (mut) plasmid circSATB2 mut. The wt and mut sequences of PEAK1 3'UTR were used to construct the PEAK1 3'UTR wt and PEAK1 3'UTR mut plasmids. A total of 293T cells were planted into the 48‐well plates, followed by co‐transfection of circSATB2 or PEAK1 3'UTR luciferase plasmid and miR‐330‐5p mimic or mimic NC. The luciferase activity detection was performed by the Dual‐luciferase Reporter Detection Kit (Promega) after transfection for 48 h.
6
Validating circRNA-miRNA Interactions
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The target binding sites were predicted using starbase (http://starbase.sysu.edu.cn). The circ_0010235 and HOXA10 sequences were cloned into the pmirGLO plasmid (Promega, Madison, WI, USA) to construct the wild-type (WT, containing the complementary sites of miR-588) plasmids WT-circ_0010235 and WT-HOXA10 3’UTR or the mutant-type (MUT, containing the mutated sites of miR-588) plasmids MUT-circ_0010235 and MUT-HOXA10 3’UTR. Luciferase activity was instantly examined using the Dual-luciferase Reporter Detection Kit (Promega) after co-transfection with each plasmid and miR-588 or miR-NC for 48 h.
7
NF-κB Pathway Modulation Assay
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To perform the NF-κB luciferase reporter assay, an NF-κB luciferase reporter plasmid and a Renilla luciferase reporter plasmid (in a 19:1 ratio) were cotransfected into RAW264.7 cells using Lipofectamine 3000 reagent (Cat# L3000015, Invitrogen). Twelve hours post transfection, RAW264.7 cells were treated with different concentrations of AaNRP (0, 0.25, 0.50, or 1.00 μg/mL) and 1.00 μg/mL LPS (Cat# HY-D1056, MCE) as a positive control for 4 h. Dual luciferase activity was detected on a Thermo Varioskan Flash platform with a dual luciferase reporter detection kit (Cat# E1910, Promega). The relative luciferase activity was calculated by normalizing the activity of NF-κB luciferase against the activity of Renilla luciferase. The NF-κB p65 nuclear translocation assay was conducted by following previously reported methods (Bartfeld et al, 2009 (link); Chen et al, 2012 (link)). In brief, RAW264.7 cells were transfected with the NF-κB p65-EGFP plasmid. Twelve hours post transfection, RAW264.7 cells were treated with 1.0 μg/mL AaNRP and 1.0 μg/mL LPS as a positive control. Four hours posttreatment, the RAW264.7 cells were fixed with 4% PFA. The translocation of NF-κB p65-EGFP was observed under a Zeiss LSM880 confocal microscope.
8
Investigating circRNA-miRNA Interactions
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The bioinformatics analysis between targets was conducted through CircInteractome (https://circinteractome.nia.nih.gov/ ) and Targetscan (http://www.targetscan.org ). The circ_0002194 wild-type (wt) sequence (with miR-637 binding sites) and the mutant-type (mut) sequence (with mutated miR-637 sites) were amplified for constructing the luciferase plasmids (circ_0002194 wt, circ_0002194 mut) using the pmirGLO plasmid (Promega, Madison, WI, USA). In addition, PACS2 three prime untranslated region (3'UTR) wt and PACS2 3'UTR mut plasmids were obtained for the binding analysis between PACS2 and miR-637. The 293 T cells (BioVector NTCC Inc., Beijing, China) were cultured in Dulbecco’s modified eagle medium containing 10% FBS, then co-transfected with wt or mut plasmids and mimic NC or miR-637 mimic for 48 h. A Dual-luciferase Reporter Detection Kit (Promega) was applied to determine the luciferase activity of each group.
9
Luciferase Assay for miRNA Regulation
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The pmirGLO plasmid (Promega) was cloned with wild‐type (WT) circ_0002360 and ZNF300 3′UTR sequences to generate WT‐circ_0002360 and WT‐ZNF300 3′UTR. The mutant‐type (MUT) plasmids MUT‐circ_0002360 and MUT‐ZNF300 3′UTR were constructed as the negative controls. The cotransfection of luciferase plasmid and miR‐NC or miR‐6751‐3p was performed for 48 h, then A549/DDP and H1299/DDP cells were harvested for luciferase intensity analysis using dual‐luciferase reporter detection kit (Promega).
10
Luciferase Assay for circRNA and 3'UTR Analysis
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The pmirGLO (Promega, Madison, WI, USA) was used as a basic luciferase vector. The original circ_0003221 or DHCR24 3’UTR sequence was considered as the wild-type (WT) sequence, which contained the miR-892b binding sites. The mutant-type (MUT) sequences were referred to circ_0003221 or DHCR24 3’UTR sequence after miR-892b sites were mutated. Then the recombinant circ_0003221 WT, circ_0003221 MUT, DHCR24 3’UTR WT and DHCR24 3’UTR MUT vectors were constructed. The 5637 and T24 cells were carried out by co-transfection with miR-892b or miR-NC and circ_0003221 vectors or DHCR24 3’UTR vectors. Cells were incubated at 37℃ for 48 hours, followed by detecting luciferase activity via Dual-luciferase Reporter Detection Kit (Promega).
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