The pGL-3 vector containing luciferase IFI35 promoter, blank control pGL-3 basic, and Renilla luciferase reporter gene vector pRL-TK-Renilla were from Jinan Weizhen Co. (Jinan, China). The Ad-IRF1 and Ad-NC cells were digested and placed in 24-well plates. After the cells adhered, pGL-3 basic, pGL-3-IFI35, and TK were transfected using Lipofectamine 3000 (Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. After transfection for 6 h, the medium was replaced. The cells were incubated at 37 °C for 48 h. The medium was removed, and the cells were washed with 100 µL of PBS twice. The 1× Passive Lysis Buffer (Dual-Luciferase Reporter Assay System E1910, Promega, Madison, WI, USA) was prepared and added (200 µL) to each well. After 20 min, the mixture was centrifuged at 13,000 rpm for 30 s, and 50 µL of the supernatant was transferred to a white opaque 96-well microplate. The pre-mixed LARII solution (30 µL) of the Promega Dual-Luciferase kit (Dual-Luciferase Reporter Assay System E1910, Promega, Madison, WI, USA) to each well. The results were read on a microplate reader (Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, USA). Finally, a BCA kit (P0011, Beyotime, Shanghai, China) was used to determine the protein concentration of each well for correction.
Dual luciferase reporter assay system e1910
Manufactured by Promega
Sourced in United States
The Dual-Luciferase Reporter Assay System (E1910) is a laboratory instrument designed for the quantitative measurement of gene expression. It utilizes two different luciferase reporter enzymes to enable the simultaneous quantification of two separate biological events within the same sample.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase kit, by Promega (2 mentions)
Bca kit, by Beyotime (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (2 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Prl cmv vector, by Promega (1 mentions)
Glomax microplate luminometer, by Promega (1 mentions)
Pgl4 vector, by Promega (1 mentions)
Mir 133b mimic, by RiboBio (1 mentions)
E coli atcc 25922, by American Type Culture Collection (1 mentions)
Hbe135 e6e7, by American Type Culture Collection (1 mentions)
Infinite m200 multiplate reader, by Tecan (1 mentions)
Protease inhibitor cocktail p8340, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 3 p0044, by Merck Group (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane 162 0115, by Bio-Rad (1 mentions)
Cell lysis buffer 9803, by Cell Signaling Technology (1 mentions)
Tnf α 300 01a, by Thermo Fisher Scientific (1 mentions)
32 protocols using dual luciferase reporter assay system e1910
1
Luciferase Assay for IFI35 Promoter
2
Luciferase Assay of IFI35 Promoter
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The pGL-3 vector containing luciferase IFI35 promoter, blank control pGL-3 basic, and Renilla luciferase reporter gene vector pRL-TK-Renilla were from Jinan Weizhen Co. (Jinan, China). The Ad-IRF1 and Ad-NC cells were digested and placed in 24-well plates. After the cells adhered, pGL-3 basic, pGL-3-IFI35, and TK were transfected using Lipofectamine 3000 (Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. After transfection for 6 h, the medium was replaced. The cells were incubated at 37 °C for 48 h. The medium was removed, and the cells were washed with 100 µL of PBS twice. The 1 × Passive Lysis Buffer (Dual-Luciferase Reporter Assay System E1910, Promega, Madison, WI, USA) was prepared and added (200 µL) to each well. After 20 min, the mixture was centrifuged at 13,000 rpm for 30 s, and 50 µL of the supernatant was transferred to a white opaque 96-well microplate. The pre-mixed LARII solution (30 µL) of the Promega Dual-Luciferase kit (Dual-Luciferase Reporter Assay System E1910, Promega, Madison, WI, USA) to each well. The results were read on a microplate reader (Varioskan LUX, Thermo Fisher Scienti c, Waltham, MA, USA). Finally, a BCA kit (P0011, Beyotime, Shanghai, China) was used to determine the protein concentration of each well for correction.
3
Targeting CCN2 3'-UTR in HSCs
4
Transcriptional Regulation of IbbHLH2 by Key Factors
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Dual-luciferase assays were carried out to measure the transactivation of activities of IbERF1, IbERF10, IbEBF2, IbPDC, and IbPGP19 on the promoter of IbbHLH2. In brief, the full-length cDNAs of IbERF1, IbERF10, IbEBF2, IbPDC, and IbPGP19 were inserted into the pGreen II 0029 62-SK vector and the promoter of IbbHLH2 was inserted into pGreen II 0800-LUC vector. Both constructs were transformed into Arabidopsis protoplasts. The ratio of LUC and REN enzyme activities was measured using an E1910 Dual-Luciferase® Reporter Assay System (Promega). For each interaction between transcription factors and promoters, three independent experiments were carried out, with three replicates in each experiment. A luciferase gene from Renilla driven by a 35S promoter in the luciferase vector acted as a positive control. Mixtures that contained each transcription factor and the empty vector 62-SK were also tested on the promoter as a control. The primers used for the dual-luciferase assay are listed in Supplementary Table S1 .
5
Luciferase Activity Assay Protocol
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The luciferase activity of samples was detected by E1910 Dual Luciferase Reporter Assay System (Promega, USA). The culture medium was discarded 48 h after transfection, and cells were washed twice with PBS. Afterwards, the cells in each well were added 100 μL of passive lysis buffer and gently shaken at room temperature for 15 min to collect the lysate. Afterwards, 20 μL of the lysate was added into GloMax microplate luminometer (Promega, USA). After measurement of the background for 2 s, 100 μL of LARII solution was added into each sample, quickly mixed and measured for 2 s. Subsequently, 100 μL of Stop and Glo reagent was added, rapidly mixed and measured for 2 s.
6
Bioinformatic Analysis of DKK2 and miR-154
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Bioinformatic analysis was performed using DIANA Tools (Athena Innovation, Greece). According to the predicted sequence of binding sites between DKK2 and miR-154, DKK2 was found to be mutated at the presumed binding site. Endothelial cells were cultured with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and co-transfected with pmir-GLO, pmir-GLO-DKK2-wt, or pmir-GLO-DK2-mut, and mir-154 mimic or microRNA-NC. After 48 hours of preparation and transfection, the relative luciferase activity of endothelial cells was measured using the E1910 Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
7
Transcriptional Regulation Assay of RhAP2 and RhAP2L
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LUC/REN activity was determined as previously described (Gao et al., 2019 (link)). The promoter fragments of RhAP2 and RhAP2L were inserted into the pGreenII0800-LUC vector as reporter constructs (Hellens et al., 2005 (link)). The coding sequence of RhMYB17 was inserted after the 35S promoter in the pGreenII62-SK vector as the effector. The recombinant plasmids were transformed into Agrobacterium (GV3101) and introduced into tobacco leaves by injection. Following 3 d of incubation, luminescence was detected with a live imaging apparatus and measured using the E1910 Dual-Luciferase Reporter Assay System (Promega) (Liang et al., 2020 (link)).
8
Constructing and Evaluating IbANS Promoter
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To construct the dual-luciferase reporter vector, the 1.9 kb upstream promoter region of IbANS (Itf13g04110.t1) (from the ATG start codon) was amplified from the genomic DNA of tuberous roots from the sweet potato cultivar ‘Xuzi No. 8’ and inserted into a pGreen II 0800-LUC binary vector. Moreover, Agrobacterium transformation and injection preparation were the same as the methods described for the transient transformation assay in tobacco leaves and strawberries.
Agrobacterium cells harboring the pGreen II 0800-LUC recombinant vector, pSAK277 vector, IbMYB340, IbbHLH2, and IbNAC56 were mixed at a 1:3:3:3 ratio. The mixture of Agrobacterium cells was injected into young N. tabacum leaves that were 2 weeks old. At 48–72 h after infiltration, the LUC and Ren activity were measured with an E1910 Dual-Luciferase® Reporter Assay System (Promega).
Agrobacterium cells harboring the pGreen II 0800-LUC recombinant vector, pSAK277 vector, IbMYB340, IbbHLH2, and IbNAC56 were mixed at a 1:3:3:3 ratio. The mixture of Agrobacterium cells was injected into young N. tabacum leaves that were 2 weeks old. At 48–72 h after infiltration, the LUC and Ren activity were measured with an E1910 Dual-Luciferase® Reporter Assay System (Promega).
9
Constructing CML5 Promoter-Driven Luciferase Reporter
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To construct the dual-luciferase reporter vector, the 2 kb upstream promoter region of CML5 (from the ATG start codon) was amplified from the genomic DNA of peels from ‘Honeycrisp’ apples and inserted into a pGreen II 0800-LUC binary vector. In addition, the injection solutions were prepared as in Section 5.10 . Agrobacterium cells harboring the pGreen II 0800-LUC recombinant vector and pSAK277 vector, ERF2/17, bHLH2 were mixed at a 1:9 ratio. The mixture of Agrobacterium cells was injected into young N. tabacum leaves that were 2 weeks old. At 48-72 h after infiltration, the LUC and Ren activity were measured with an E1910 Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA).
10
VEGF mRNA Regulation by IMP3
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VEGF 5’‐UTR, CDS sequence and 3’‐UTR were amplified and inserted into a pGL4 vector (Promega, USA), and named pGL4‐5’‐UTR, pGL4‐CDS and pGL4‐3’‐UTR, respectively. 293T cells were seeded onto 24‐well plates 24 h before the experiment. Empty vector, pGL4‐3’‐UTR, pGL4‐CDS or pGL4‐5’‐UTR together with AAV2‐IMP3 or AAV2‐Control and Renilla luciferase plasmid were co‐transfected into 293T cells by Lipofectamine 3000 reagent (Invitrogen, USA). Forty‐eight hours post‐transfection, the cells were harvested and lysed. Firefly luciferase activities were detected with a Dual‐Luciferase Reporter Assay System E1910 (Promega, USA) and normalized to control Renilla luciferase levels.
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