HeLa cells were seeded at a density of 5 × 104 cells per well in 24-well plates. After 24h, the cells were co-transfected with 500ng pmiR-FAM129A-3’UTR-wild or pmiR-FAM129A-3’UTR-mut plasmids and 100nM hsa-miR-4521 mimics or mimics negative control (mimics NC) or inhibitor or inhibitor negative control (inhibitor NC) using the Lipofectamine 3000 Reagent. Forty-eight hours after transfection, luciferase assays were measured using Dual-GLO® Luciferase Assay System Kits (Promega, USA) following the manufacturer’s instructions by a Fluorescence/Multi-Detection Microplate Reader (Biotek, USA). The relative luciferase activity was calculated by calculating the ratio of renilla luciferase activity to firefly luciferase activity.
Dual glo luciferase assay system kit
Manufactured by Promega
Sourced in United States
The Dual-GLO Luciferase Assay System Kit is a laboratory tool designed to quantify the activity of firefly and Renilla luciferase reporter genes. The kit provides reagents for the sequential measurement of both luciferase activities in a single sample, allowing for the normalization of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence multi detection microplate reader, by Agilent Technologies (36 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Pgl3 vector, by Promega (7 mentions)
Glomax microplate luminometer, by Promega (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Eif4g2 3 utr, by Genechem (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Pmirglo vector, by Promega (3 mentions)
Multifunction microplate reader, by Agilent Technologies (3 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (3 mentions)
Prl tk, by Promega (3 mentions)
Pgl3 basic vector, by Promega (3 mentions)
Prl tk renilla luciferase plasmid, by Promega (3 mentions)
Prl tk vector, by Promega (2 mentions)
Fugene 6, by Promega (2 mentions)
Glomax 96 microplate luminometer, by Promega (2 mentions)
X tremegene hp dna transfection reagent, by Merck Group (2 mentions)
Schneider s insect medium, by Merck Group (2 mentions)
Infinite m200 pro plate reader, by Tecan (2 mentions)
126 protocols using dual glo luciferase assay system kit
1
Luciferase Assay for miR-4521 Targeting
2
Regulation of DDX6 by miR-152-3p
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The binding site between miR-152-3p and DDX6 was predicted from the starBase. Wild-type (WT) and mutated (MUT) fragments of DDX6 3ʹUTR containing potential binding site with miR-152-3p were synthesized and respectively subcloned into pmirGLO vectors (Promega) to construct pmirGLO-DDX6-WT/MUT reporters. Phusion Site-Directed Mutagenesis Kits (Thermo Fisher) were applied to mutate the predicted binding site. The above reporters were cotransfected with miR-152-3p inhibitor or NC inhibitor into HUVECs using Lipofectamine 3000 reagent (Invitrogen). The luciferase activities of DDX6-WT/MUT were determined utilizing Dual-Glo® Luciferase Assay System Kits (Promega) after 48 hours of cotransfection [31 (link)].
3
Dual-GLO® Luciferase Assay of MDA5-3'UTR
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Luciferase activity was measured using Dual-GLO® Luciferase Assay System Kits (Promega, Madison, WI, USA) following the manufacturer's instructions. DF-1 cells were seeded at a density of 1 × 103 cells per well in 96-well plates. After 24 h, the cells were co-transfected with 100 ng pmir-GLO- WT-MDA5-3′ UTR- (wild-type) or pmir-GLO-MT-MDA5-3′ UTR(mutant-type) plasmids, or 100 nM gga-miR-34b-5p mimic and miR-NC using the Lipofectamine 3000 Reagent. Fourty-eight hours after transfection, luciferase assays were performed using a Fluorescence/Multi-Detection Microplate Reader (Synergy 2, Biotek, Winooski, VT, USA). The values obtained were normalized to the levels of a Renilla luciferase plasmid (pRL-TK Vector) levels.
4
Characterizing miR-27a-3p Regulation of MMP2
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The 5'untranslated region (UTR) of MMP2 and its mutated 5'UTR were cloned into GP-Check2 plasmids (GenePharma, Shanghai), named GP-Check2-MMP2 and GP-Check2-mut-MMP2, respectively. Mock plasmids GP-Check2-Mock was used as negative control. 1.5×104 WS1 cells were transfected with GP-Check2-MMP2, GP-Check2-mut-MMP2 and GP-Check2-Mock constructs, respectively, using lipofectamine® 2000 (Life Technologies, USA). Twenty-four hours later, miR-27a-3p mimics or its negative control (NC) was transfected into the WS1 cells. Luciferase activity was measured on a EnSpire plate reader (Synergy 2, Bio-Tek, USA) 24 h post transfection using a Dual-Glo® luciferase assay system kit (Promega) according to the manufacturer's protocol.
5
Mapping Promoter Region of chCH25H
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To identify the promoter region of chCH25H, the 2077 bp nucleotide sequences before the CH25H coding region were truncated into 6 segments to construct different dual luciferase reporter vectors. The first nucleotide before the chCH25H coding region is numbered −1, and the different dual luciferase reporter vectors are named PGL3- p2077(−2077/−1), p1000 (−1000/−1), p500 (−500/−1), p250 (−250/−1), p125 (−125/−1) and p75 (−75/−1), respectively (Figure 4A). The primers used to construct PGL3 plasmids were showed in Supplementary Table S1 .
The PGL3 plasmids (p2077, p1000, p500, p250, p125, p75) were co-transfected with pRL-TK in DF1 cells, respectively. The pGL3-basic was co-transfected with pRL-TK as a control. Firefly and Renilla luciferase activities were measured at 24 h post-transfection using a Dual-GLO Luciferase Assay System Kit (Promega, Madison, WI, USA). Luminescence was measured using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Shoreline, WA, USA). Firefly luciferase activities were normalized to Renilla luminescence in each well.
Furthermore, the common interferon stimulated response elements (ISRE) consensus 5’ A/GGTTTCN(1–2)TTTCC/T3’ or its reverse complement, and the common gamma activated sequence (GAS) consensus 5’ TTNCNNNAA3’ were screened at upstream of chCH25H according to previous studies [23 (link)].
The PGL3 plasmids (p2077, p1000, p500, p250, p125, p75) were co-transfected with pRL-TK in DF1 cells, respectively. The pGL3-basic was co-transfected with pRL-TK as a control. Firefly and Renilla luciferase activities were measured at 24 h post-transfection using a Dual-GLO Luciferase Assay System Kit (Promega, Madison, WI, USA). Luminescence was measured using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Shoreline, WA, USA). Firefly luciferase activities were normalized to Renilla luminescence in each well.
Furthermore, the common interferon stimulated response elements (ISRE) consensus 5’ A/GGTTTCN(1–2)TTTCC/T3’ or its reverse complement, and the common gamma activated sequence (GAS) consensus 5’ TTNCNNNAA3’ were screened at upstream of chCH25H according to previous studies [23 (link)].
6
Regulation of IRS1 by lncIRS1 and miR-15
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DF‐1 cells were seeded in 96‐well plates 1 day before transfection and then co‐transfected with (I) pmirGLO‐IRS1 reporter, (II) pmirGLO‐site 1 of lncIRS1 reporter, (III) pmirGLO‐site 2 of lncIRS1 reporter, and (IV) pmirGLO‐site 3 of lncIRS1 reporter, and miRNA mimics or mimic NC using Lipofectamine 3000 reagent. After 48 h, luciferase activity analysis was performed using a Fluorescence/Multi‐Detection Microplate Reader (BioTek, Winooski, VT, USA) and a Dual‐GLO Luciferase Assay System Kit (Promega, USA). The firefly luciferase activities were normalized to Renilla luminescence in each cell‐well.
RNA immunoprecipitation used the Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) and the anti‐Argonaute2 (AGO2) antibody (Abcam, Cambridge, UK) according to the manufacture's protocol. After the antibody was recovered by protein A + G beads, qRT‐PCR was performed to detect IRS1, lncIRS1, and miR‐15 family in the precipitates.
RNA immunoprecipitation used the Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) and the anti‐Argonaute2 (AGO2) antibody (Abcam, Cambridge, UK) according to the manufacture's protocol. After the antibody was recovered by protein A + G beads, qRT‐PCR was performed to detect IRS1, lncIRS1, and miR‐15 family in the precipitates.
7
Wnt3a Signaling Pathway Regulation
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Wnt3a, BMS345541, DAPI, and MTT were from Sigma‐Aldrich (Indianapolis, IN, USA). Fetal calf serum and RPMI‐1640 medium were purchased from Gibco (Grand Island, NY, USA). Lipofectamine RNAi MAX, Lipofectamine 2000, stealth‐siRNA, goat anti‐mouse and anti‐rabbit secondary antibodies conjugated to HRP, and donkey anti‐rabbit antibody–Alexa Fluor 647 were purchased from Invitrogen (Carlsbad, CA, USA). Monoclonal antibodies against cyclin A, cyclin B1, cyclin D1, cyclin E, p21, β‐actin, and polyclonal antibody against RXRα were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies against β‐catenin and phosphorylated β‐catenin were purchased from Cell Signaling Technology (Danvers, MA, USA). The PVDF membranes were obtained from Millipore (Billerica, MA, USA) and the 5‐ethynyl‐2′‐deoxyuridine (EdU) assay kit was from RiboBio (Guangzhou, China). The Dual‐Glo Luciferase Assay System kit was purchased from Promega (Madison, WI, USA) and the EliVision Plus kit was from Maixin Bio (Fuzhou, China).
8
Dual-Glo Luciferase Assay Normalization
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At 24 h post-transfection, Renilla and firefly luciferase activities were measured by luminometer in the same plate using the Dual-Glo Luciferase Assay System kit (Promega); 2030 ARVO X5 (Perkin Elmer). Renilla luciferase activity values were normalized for transfection efficiency using the corresponding firefly luciferase activity as an internal control. Three independent transfection experiments were performed, each in triplicate.
9
Centyrin-siRNA Conjugate Luciferase Assay
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To test the activity of Centyrin-siRNA conjugates against targets that are not expressed endogenously in the cell lines used, a luciferase reporter system was used. Cells were plated in 96-well plates (3,000 cells per well) for 4 h, then transfected using Lipofectamine 2000 according to manufacturer’s instructions with the Multi-site psiCheck-2 Dual Luciferase Reporter Plasmid (Promega, Madison, WI, USA) containing the target gene of interest. Cells were thoroughly rinsed 24 h later to remove excess transfection agent and treated with Centyrin-siRNA conjugates for 72 h. Cells were assayed for both Renilla and firefly luciferase expression using the Dual-Glo Luciferase Assay System kit (Promega), essentially according to manufacturer’s instructions (manufacturer’s supplied Luciferase assay Reagent II was diluted 1:1 with complete media prior to use). Luminescence was detected using an Envision multi-mode plate reader (Perkin Elmer, Waltham, MA, USA). Background signal from untransfected cells was subtracted from each assay well, and Renilla expression was normalized to the nonspecific Firefly expression.
10
Dual-GLO Luciferase Assay of PRRX1 3'UTR
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After co-transfection of pmirGLO-PRRX1-3′UTR-WT or pmirGLO-PRRX1-3′UTR-MT with gga-mir-133a-3p mimic or mimic NC for 48 h, respectively, firefly and Renilla luciferase activities were detected using a Dual-GLO Luciferase Assay System Kit (Promega, United States) according its instruction. Multi-function microplate reader (Biotek, United States) was used to detect the firefly luciferase and Renilla luminescence activities.
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