Putative genes that are miR-124 targets were analyzed using TargetScan V7.1 (http://www.targetscan.org/vert_71/ ). To generate the luciferase reporter vector, a 312 bp human Stat3 gene 3′-untranslated regions (3′-UTR) segment encompassing the predicted miR-124 binding sites was PCR-amplified and subcloned into the pGL3 luciferase plasmid. The mutant construct was made with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Dual luciferase activity assay was performed according to the instruction manual (Promega, Madison, WI, USA).
Dual luciferase activity assay
Manufactured by Promega
Sourced in United States
The Dual Luciferase Activity Assay is a laboratory tool that measures the activity of two different luciferase enzymes simultaneously in the same sample. It provides a rapid and sensitive method for quantifying gene expression or evaluating the activity of regulatory elements.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lumat lb 9507 ultra sensitive tube luminometer, by Berthold Technologies (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Prl cmv, by Promega (2 mentions)
Glomax 20 20 luminometer, by Promega (2 mentions)
Prl tk, by Takara Bio (1 mentions)
Pmirglo, by Promega (1 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Mimic nc, by RiboBio (1 mentions)
Inhibitor nc, by RiboBio (1 mentions)
Pgl3 vector, by Promega (1 mentions)
Sybr green dye, by Takara Bio (1 mentions)
Psicheck 2, by Thermo Fisher Scientific (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Psicheck 2, by GenePharma (1 mentions)
20 protocols using dual luciferase activity assay
1
Validating miR-124 Target Genes
2
MiR-548ag Regulates DNMT3B 3'UTR
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The TargetScan database (http://www.targetscan.org ) was used for analyzing the 3’UTR binding sites of miR-548ag and DNMT3B. The Luciferase plasmids were constructed at and provided by Shanghai GenePharma: hDNMT3B-mir-548ag wt (5’-TTTAGGCTGAAAGATGACGGATGCCTAGAGTTTACCTTATGTTTAATTAAAATCAGTATTTGTCT-3’) and hDNMT3B-mir-548ag mut (5’-TTTAGGCTGAAAGATGACGGATGCCTAGAGTaatggaaATGTTTAATTAAAATCAGTATTTGTCT-3’). The dual luciferase activity assay was performed according to the manufacturer’s instructions (Promega).
3
Dual Luciferase Reporter Assay in DF-1 Cells
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Dual luciferase reporter assay was comprised of two reporters. One is Renilla luciferase and the other is a firefly luciferase in pmirGLO containing the assayed 3′-UTR sequence. For luciferase reporter assay, DF-1 cells were plated 1 day before transfection. On the 2nd day, 200 ng of luciferase reporter plasmid and 10 pmol of the indicated RNA oligonucleotides were also transfected into DF-1 using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). The cells were collected at 48 h of post-transfection, and the dual-luciferase activity assay was performed according to the manufacturer’s instructions (Promega, Medison, WI, USA). Luciferase activity was detected using a Lumat LB 9507 Ultra-Sensitive Tube Luminometer (Titertek Berthold, Nanjing, China). Firefly luciferase activity of each sample was normalized by Renilla luciferase activity. All transfection experiments were performed in triplicate and repeated at least three times.
4
Regulation of RP11-551L14.4 by miR-4472
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The binding sites of RP11-551L14.4 and miR-4472 were predicted with the LncBase Predicted v.2 database. The RP11-551L14.4-wild-type (RP11-551L14.4-WT) luciferase reporter gene plasmid and the RP11-551L14.4-mutant (RP11-551L14.4-MUT) reporter gene plasmid were constructed by GenePharma (Shanghai, China). T47D cells were co-transfected with miR-4472 mimic or miR-NC, and with RP11-551L14.4-WT or RP11-551L14.4-MUT reporter gene plasmid using Lipofectamine 3000 reagent (Invitrogen). After incubated for 48 h, the intensity of the fluorescence was measured using dual-luciferase activity assay according to the manufacturer protocol (Promega, Madison, WI, USA). The following sequences were used: miR-4472 mimic, 5′-GGUGGGGGGUGUUGUUUU-3′; miR-NC mimic, 5 ′-UUGUCCGAACGUGUCACGU-3′.
5
Luciferase Assay for YWHAZ 3'UTR
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DF-1 cells in 24-well plates were co-transfected with 200 ng of psiCHECK-2–YWHAZ-3′UTR (wild-type and mutant) and 10 pmol of the indicated RNA oligonucleotides using Lipofectamine 3000 (Invitrogen). The cells were collected at 48 h post-transfection, and the dual-luciferase activity assay was performed according to the manufacturer’s instructions (Promega). Luciferase activity was detected using a Lumat LB 9507 Ultra-Sensitive Tube Luminometer (Titertek Berthold, Nanjing, China). All transfection experiments were performed in triplicate and repeated at least three times.
6
Validating TSLP as a miR-19b Target
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The MicroRNA.org website was used to verify whether TSLP was a direct target of miR-19b. The full length TSLP gene 3’UTR was cloned into multiple cloning sites downstream of the pmirGLO (Promega Company) expression vector luciferase gene. This 3’UTR was also used to computationally predict miR-19b binding sites. Renilla luciferase in the pRL-TK vector (TaKaRa Company) was used as an internal reference to adjust for cell number differences and assess transfection efficiency. miR-19b mimics and NC were transfected into HEK-293T cells with wild-type (WT) and mutant (MUT) luciferase reporter vectors, respectively. After 48 h of transfection, cells were collected and split, and the dual luciferase activity assay was performed according to the manufacturer's instructions (Promega, Madison, Wisconsin, USA).
7
Influenza Polymerase Activity Assay
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293T cells were cotransfected with plasmids that express PB1, PB2, PA, or NP of the PR8 H1N1 influenza A virus and a plasmid containing polymerase I promoter-driven negative-sense open reading frame of firefly luciferase. A polymerase II promoter-driven Renilla luciferase reporter plasmid was also cotransfected into the cells as the transfection efficiency control. At 24 h posttransfection, cell lysates were collected, and the relative luciferase activity was measured by dual luciferase activity assay (Promega) as described previously (Chang et al., 2017 ). The same approach was used to cotransfected 293T cells with the PYCR2-expressing plasmid, the influenza polymerase subunits, and the luciferase reporters to determine the role of PYCR2 in viral polymerase activity.
8
Validating miR-186 and SMAD6 Interaction
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Dual luciferase reporter gene assay was used to detect whether SMAD6 was the direct target gene of miR-186. The full-length of the 3'-untranslated region (UTR) region in SMAD6 gene was cloned and amplified. Polymerase chain reaction (PCR) products were cloned into multiple sites of luciferase reverse primer of pmirGLO Dual-Luciferase miRNA Target Expression Vector (E1330; Promega Corporation, Madison, Wisconsin). Following this, the bioinformatics software was used to predict the site-directed mutagenesis of miR-186 and target gene binding sites and constructed the wild plants containing SMAD6 3'-UTR with miR-186 target site, which was named SMAD6-wild-type (Wt). Afterwards, the SMAD6 3'-UTR and miR-186 complementary sites were mutated, and the luciferase plasmid containing S mutant sequences was constructed, named as SMAD6-Mutant (Mut). The pRL-TK carrier (E2241; Takara Biotechnology Ltd., Liaoning, China) with expression of Renilla luciferase was regarded as the internal reference and was used to adjust the difference of transfection efficiency and cell number. The mice from the miR-186 and NC groups were respectively transfected into cells of fracture tissue. The dual luciferase activity assay was used in accordance with the method provided by Promega Corporation.
9
Pancreatic Cancer Cell Line Profiling
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Pancreatic cancer cell lines (PANC-1, AsPC-1, BxPC-3, and HPAC) were purchased from the Shanxi Academy of Medical Sciences (Taiyuan, China). Normal human pancreatic epithelial cells (HPDE 6-C7 and HEK293T) were purchased from the Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), and Opti-MEM medium were procured from Gibco (Grand Island, NY, USA). Lipofectamine 2000 was procured from Invitrogen (Carlsbad, CA, USA). The PrimeScript TM RT Kit and SYBR Green dye were procured from TaKaRa (Otsu, Shiga, Japan). Mimic-miR-203-3p, mimic-NC, inhibitor-miR-203-3p, and inhibitor-NC were obtained from RiboBio (Guangzhou, China). The pGL3 vector and Dual-Luciferase Activity Assay were purchased from Promega (Madison, WI, USA). The EdU-Alexa Fluor ® 488 Cell Proliferation Assay was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Double-Staining Cell Apoptosis Assay was purchased from Nanjing Kaiji (Nanjing, China).
10
Analysis of IRS1 3'UTR-miR-487b-3p interaction
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The 3'UTR of goat IRS1 mRNA were ampli ed from myoblast cells cDNA with the primers as shown in table 2. The wild type or mutant 3'UTR sequences of IRS1 were cloned into the psi-CHECK2 vector (Promega, Germany) with restriction sites of Xho I and Not I, respectively. HEK293T cells were seeded in 48-well culture plates at a density of 1×10 4 cells per well. The cells were co-transfected with 250 ng of the wild-type (WT, psi-CHECK2-IRS1-3'UTR) or the mutant (Mutant, psi-CHECK2-IRS1-3'UTR-mut) 3'UTR plasmids and 100 nM miR-487b-3p mimics or negative control (NC) using the Lipofectamine 3000 reagent (L3000015, Invitrogen, Waltham, MA) following the manufacturers protocol. After the transfection for 48 hours, cells were harvested and lysed in passive lysis buffer (Promega, USA), and the dualluciferase activity assay was performed according to the manufacturer's instruction (Promega, USA).
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