For the measurement of the effects of AcERFs on the transcription of AcKUP2, the ORF of AcERFs were cloned into the pGreenII 0029 62–SK vector driven by the 35S promoter as an effector, and promoter sequences of AcKUP2 were inserted into the pGreenII 0800–LUC vector as reporter. All the constructs were transformed in A. tumefaciens and, then, injected into tobacco leaves according to the method of subcellular localisation. LUC and REN luciferase activity were measured using a dual-luciferase assay kit (YEASEN, Shanghai, China) according to the manufacturer recommendation. The LUC to REN ratio was calculated. At least six biological replicates were performed per assay.
Dual luciferase assay kit
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The Dual-luciferase assay kit is a laboratory instrument designed to measure and analyze the activity of two different luciferase reporter enzymes simultaneously. It provides a reliable and efficient method for studying gene expression and cellular processes.
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12 protocols using dual luciferase assay kit
1
AcERFs Regulate AcKUP2 Transcription
2
Cell-based Assay for Ligand-Receptor Binding and Transactivation
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All plasmids were constructed by XinJia Medical Technology (Nanjing, China). The gene sequences for the ligand-binding domain (LBD) of RARα and RXRα were inserted into the pBIND plasmid to construct the pBIND-based Gal4-RARα LBD (RARα-LBD) and Gal4-RXRα LBD (RXRα-LBD) vectors. These plasmids, along with pGL4.35[luc2P/9XGAL4UAS/Hygro], were co-transfected into HEK293T cells for cell-based binding activity assays. pGL4.35-based luc2P-4XDR1 and luc2P-4XDR5, along with pBIND-based RXRα or RARα expression plasmids, were co-transfected into HEK293T cells for the transactivation assay of DR1-or DR5-triggered reporter gene expression.
HEK293T cells were seeded in 6-well plates for 12 h and then transfected with 2 μg of the above plasmids (Equimass) using Lipofectamine 3000 and P3000 according to the manufacturer's instructions. After 12 h of transfection, the cells were digested and divided into 96-well plates. Transfected cells were then incubated for an additional 12 h before treatment with the test agent. After 24 h of drug treatment, the cells were lysed and assayed for luciferase activity according to the instructions of the dual luciferase assay kit (Yeasen, Shanghai, China). The transfection efficiency was normalized to Renilla luciferase activity.
HEK293T cells were seeded in 6-well plates for 12 h and then transfected with 2 μg of the above plasmids (Equimass) using Lipofectamine 3000 and P3000 according to the manufacturer's instructions. After 12 h of transfection, the cells were digested and divided into 96-well plates. Transfected cells were then incubated for an additional 12 h before treatment with the test agent. After 24 h of drug treatment, the cells were lysed and assayed for luciferase activity according to the instructions of the dual luciferase assay kit (Yeasen, Shanghai, China). The transfection efficiency was normalized to Renilla luciferase activity.
3
Transcriptional Regulation of Stress Responses
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The transcriptional activity of CsWRKY25 was analyzed with dual-luciferase assay system. The vector pBD-CsWRKY25 and pEAQ-CsWRKY25 was built as the effectors, and promoters of CsRbohB, CsRbohD and CsPR10 were cloned into pGreenII 0800-LUC vector as reporters, respectively. The reporter and effector were cotransformed into tobacco leaves to measure the LUC/REN ratio using a dual-luciferase assay kit (Cat No.11402, Yeason, Shanghai, China) at 2–3 days after infiltration (Sainsbury et al., 2009 (link); Fan et al., 2018a (link); Wang et al., 2021 (link)). At least six independent repeats were set.
4
Validation of miR-6888 Binding to HOTAIR and SYK
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Potential binding sites of miR-6888 in HOTAIR or SYK were predicted using the shared site. The sequences containing wild-type (WT) and mutated (MUT) HOTAIR or SYK 3’-UTR were synthesized from GenePharma (USA) and subcloned into the pMIR-REPORTTM vector (Thermo Fisher Scientific) to construct a luciferase reporter vector. HOS and Saos2 cells were transfected with an miR-6888 mimic and WT or MUT, respectively. Forty-eight hours after transfection, the dual-luciferase assay kit (Yeasen, China) was used to determine the luciferase activity.
5
Ccn2 3'-UTR Interaction Validation
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The predicted 3′-UTR sequence of Ccn2 interacting with miR-15b-5p/miR-290a-5p and mutated sequences within the predicted target sites were synthesized and inserted into the pmirGLO control vector (Promega). The insert sequences used for the luciferase reporter assay are listed in Supplementary Table 10 . HEK293T cells were transfected with 50 nM miR-15b-5p/miR-290a-5p or negative control and 400 ng of the wild-type or mutant 3′UTR plasmid by Lipofectamine 2000 (Invitrogen). After 48 h of transfection, the luciferase activity of the cells was measured using a Dual Luciferase Assay Kit (Yeasen). Renilla luciferase was used to normalize the value of firefly luciferase.
6
Analysis of HES1 and HES5 Promoter Activity
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Luciferase reporter constructs containing the HES1 and HES5 promoters and 8× RBPJ-binding sites were generated by inserting the HES1 and HES5 promoters and the 8× RBPJ binding site sequence into the pGL3 basic luciferase vector upstream of the firefly luciferase gene. For the luciferase assay, HEK293T cells were plated at 50% confluency in 24-well plates and grown overnight. The firefly luciferase reporter construct and the Renilla control reporter were cotransfected into the cells at a molar ratio of 10:1. After 24 hours of culture, the luciferase activity was assayed with the Dual Luciferase Assay Kit (11402ES60, YEASEN) with normalization to Renilla activity.
7
Characterization of SPHK1 Promoter Binding
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In hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget/#!/prediction ), the binding site of ATF3 and the SPHK1 promoter was predicted. The WT and mutant (MUT) binding sequences of the SPHK1 promoter to ATF3 were sub‐cloned into the pGL3‐basic plasmid to construct a luciferase reporter gene vector. After 24 h, the luciferase activity of the cells was assessed using the Dual Luciferase Assay Kit (11402ES60, Yeasen).
8
NF-kB Transcriptional Activity Modulation
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HUVECs at a density of 104 cells/well were seeded into 96-well plates and were then co-transfected with 100 ng NF-kB luciferase reporter plasmid (cat. no. YB003B; Shanghai Yu Bo Biological Technology Co., Ltd.) and 10 ng pGMLR-CMV luciferase Reporter plasmid (cat. no. 11558ES03; Shanghai Yeasen Biotechnology, Co., Ltd.) using Lipofectamine® 3000 (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h post-transfection, cells were stimulated with 100 ng/ml LPS with or without tyrosol (0.5 mM or 1 mM) at 37˚C for 24 h. Luciferase activity was measured using the corresponding Dual-Luciferase Assay kit (Shanghai Yeasen Biotechnology, Co., Ltd.). Renilla luciferase was used as internal reference.
9
Luciferase Assay of miR-545-3p Targets
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The wide-type (WT) complementary sequence of FAM83H-AS1 or 3ʹUTR HS6ST2 to miR-545-3p and the matched mutant (MUT) sequence were amplified and ligated into pmirGLO vectors to produce FAM83H-AS1-WT, FAM83H-AS1-MUT, 3ʹUTR HS6ST2-WT and 3ʹUTR HS6ST2-MUT luciferase vectors. These constructed vectors were accompanied by miR-545-3p mimic or mimic NC to transfect HCC827 and NCI-H1650 cells. Finally, the relative luciferase activities were measured after 48 h of transfection using a Dual-Luciferase Assay Kit (Yeasen, China) [39 (link)].
10
Elucidating Transcriptional Regulation Mechanisms
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The promoters of NaWRKY70 and NaF6ʹH1 were amplified with specific primers and cloned into the pCAMBIA3301-LUC-REN reporter vector. The coding sequences of NaEIN3-like1 and NaWRKY70 were amplified with specific primers and inserted into the pCAMBIA3301 effector vector. The constructed reporter and effector vectors were transformed into Agrobacterium tumefaciens GV3101 strain, and transiently expressed in N. benthamiana leaves. Luciferase (LUC) and Renilla luciferase (REN) activities were measured using a dual luciferase assay kit (Yeasen Biotechnology). The LUC/REN ratio was used to determine the transcriptional activity of the promoter. Supplementary Table S1 lists the primers used. All experiments were performed with at least five biological replicates.
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