The cDNA sequences of OfbHLH35 and OfERF2 were ligated into the pHBT vector (effectors). The promoter regions of CCD4 (1.06 kb), CCD1 (1.82 kb), and TPS2 (1.53 kb) were inserted into the pGreenII 0800-Luc vector (reporters). The resulting vectors were transformed into A. tumefaciens strain EHA105. EHA105 strains containing effectors and reporters were cotransformed into 5–6-week-old N. benthamiana leaves. The infiltrated plants were maintained in the incubator under continuous lighting for 72 h at 23°C. The LUC and REN activities were analyzed using a dual-luciferase assay kit (Solarbio, China). LUC/REN ratios were used to represent the relative activities of the gene promoters.
Dual luciferase assay kit
Manufactured by Solarbio
Sourced in China
The Dual Luciferase Assay Kit is a laboratory equipment designed for the simultaneous measurement of Firefly and Renilla luciferase activities in a single sample. The kit provides the necessary reagents and protocols to perform quantitative analysis of gene expression and reporter systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Pgl3 vector, by Promega (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Pmirglo, by Promega (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Glomax 20 20, by Promega (1 mentions)
Psicheck2 vector, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
17 protocols using dual luciferase assay kit
1
Transcriptional Regulation Assay in Nicotiana
2
CircRNA DOCK1 Regulatory Mechanisms
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The binding sites of circRNA DOCK1 to miR-654-5p and miR-654-5p to SMAD2 were analyzed using the Encyclopedia of RNA Interactomes (ENCORI) database (http://starbase.sysu.edu.cn/index.php ). Hep3b cells (1 ml cell suspension/well; 5×102 cells) seeded into 6-well plates were co-transfected with mimic-NC (50 nM) or miR-654-5p mimic (50 nM) and DOCK1 wild-type (WT)/mutant (MT) (50 nM) or SMAD2 WT/MT (50 nM) with Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The mutated 3′-UTR was generated using a site directed mutagenesis kit (Agilent Technologies, Inc.). At 48 h post-transfection, luciferase activities were measured using a dual-luciferase assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Renilla luminescence was used as the internal reference.
3
Evaluation of miR-107 Regulation
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The sequences of AFAP1-AS1 or PDK4 3′-UTR containing wild-type (WT) or mutant (MUT) binding sites of miR-107 were cloned into the pmirGLO vector (Promega, Madison, WI, USA) to form AFAP1-AS1-WT, AFAP1-AS1-MUT, PDK4-WT or PDK4-MUT, respectively. Next, COV504 and SKOV3 cells were co-transfected with the corresponding luciferase reporter and miR-107 or miR-NC. Then the cells were cultured for 48 h, and then a Dual-Luciferase Assay Kit (Solarbio, Beijin, China) was adopted to examine the luciferase activity of the cells in each group.
4
Verification of miRNA-lncRNA Interactions
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The interaction between miR-146b-5p and SNHG7 or Robo1 was predicted by starBase v2.0 (http://starbase.sysu.edu.cn/ ) and TargetScan (http://www.targetscan.org/vert_72/ ) online databases. The wild-type (WT) and mutant (MUT) fragments of SNHG7 were amplified and then inserted into pGL3 vector (Promega Corporation) to construct the luciferase reporter, referred to as SNHG7 WT and SNHG7 MUT, respectively. The luciferase reporter (0.1 µg) and miR-146b-5p mimics (40 nM) or miR-control (40 nM) were co-transfected into SW1990 and AsPC-1 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The construction of the luciferase reporter Robo1 3'untranslated region (UTR) WT (GUUCUCA) or Robo1 3'UTR MUT (ACCAAUG) were similar to that of SNHG7 WT (CAGTTCTC) or SNHG7 MUT (ATCAAGCA) reporter. The luciferase activity was assessed using a dual-luciferase assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Renilla luciferase activities were used as the internal control for the normalization of firefly luciferase activity.
5
Measuring miR-224 and TLR2 Luciferase Activity
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The target gene analysis of miR‐224 and TLR2 was performed using a biological prediction site miRmap. The cells were seeded into a 6‐well plate at a density of 2 × 105 per well. After the cells were attached to the wells, transfection was carried out according to the above method. After successful transfection, the cells were incubated for 48 hours and then collected. The luciferase activity of miR‐224 and TLR2 in cells was measured according to the manufacturer's instructions in Genecopoeia's dual luciferase assay kit (D0010, Beijing Solarbio Science & Technology Co., Ltd.). Fluorescence intensity was measured using a Promega Glomax 20/20 luminometer fluorescence detector (E5311, Shaanxi Zhongmei Biotechnology Co., Ltd.).
6
Dual Luciferase Assay for Transfection Evaluation
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Cells from each group were seeded into 6-well plates at a concentration of 2 × 105 cells/well and subjected to transfection. After 48 h of transfection, cells were collected, and luciferase activities were detected using dual luciferase assay kit (D0010, Solarbio, Beijing, China) according to the manufacturer’s instruction. The luciferase activated reflected by luminescence was determined with Glomax20/20 luminometer (E5311, Promega, Madison, WI, USA).
7
Dual-Luciferase Assay of MIAT-miR-378a-5p Interaction
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The wild-type sequence (WT; 5′-AAACCUGGCAGAUGGUCCUAGGUCAGGAU-3′) and mutant sequence (MUT; 5′-AAACCUGCUCGGUAAUGACAUCAGGCAGU-3′) of MIAT specifically synthesized in combination with miR-378a-5p were inserted into the dual-luciferase reporter vector pmirGLO (Promega, Madison, WI, USA) to construct dual-luciferase reporter plasmids (pmirGLO-MIAT-WT and pmirGLO-MIAT-MUT). In this experiment, SK-BR-3 and MDA-MB-231 cells (5 × 105 cells/well) were cultured in the six-well plate. About 5 µg pmirGLO-MIAT-WT or pmirGLO-MIAT-MUT, 100 nM mimic control (Blank) or miR-378a-5p mimic, and Lipofectamine 3000 reagent were diluted with Opti-MEM medium, respectively. Then, the dilutions were mixed and maintained at room temperature for 10 min. After that, these lipid complexes were added to incubate the SK-BR-3 and MDA-MB-231 cells at 37℃ for 48 h. The activity of luciferase was tested on the dual-luciferase reporter system (30IOC; Promega) using a dual-luciferase assay kit (D0010; Solarbio, Beijing, China).
8
Predicting LncRNA-miRNA-mRNA Interactions
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The StarBase database (http://starbase.sysu.edu.cn/ ) was used to predict the binding sites among lncRNA NUTM2A-AS1, miR-376a-3p and YAP1. For dual luciferase reporting assay, NUTM2A-AS1-WT, NUTM2A-AS1-MUT, YAP1-WT or YAP1-MUT were co-transfected into U251 cells with miR-376a-3p mimic or mimic control. After 48 h of transfection, cells were collected and luciferase activity was assessed using a dual luciferase assay kit (Solarbio).
9
Luciferase Reporter Assay for miRNA Targets
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The wild-type (WT) sequence of circCEP128 or SDC1 3ʹ UTR with miR-515-5p complementary site was cloned into pGL3-Basic vector (Promega) to generate the luciferase reporter vectors (circCEP128 WT or SDC1 3ʹUTR WT). The mutant (MUT) vectors (circCEP128 MUT and SDC1 3ʹUTR MUT) were also constructed via mutating corresponding binding site of miR-515-5p. T24 and 5637 cells were transfected with 1 μg of vector and 30 nM of miR-515-5p mimic or miR-NC. The luciferase activities were examined via a dual-luciferase assay kit (Solarbio) with Renilla luciferase activity as the normalization control.
10
Luciferase Assay for circRNA 3'UTR Targeting
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The wild‐type and mutant‐type sequences of circRUNX1 or RAB3D 3'UTR containing miR‐760 binding sites or mutation sites were cloned into pGL3 reporter vector to produce the circRUNX1 WT/MUT and RAB3D 3'UTR WT/MUT vectors. H1975 and PC9 cells were seeded in 24‐well plates and were then cotransfected with the above vectors and miR‐760 mimic/miR‐NC. Relative luciferase activities (Firefly/Renilla) were measured by dual‐luciferase assay kit (Solarbio).
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