DU145 and LNCaP cells were cultured in 96-well plates for 24 h in prior of being co-transfected with dual-luciferase reporter vector and miR-615-5p mimic or miRNA NC using Lipofectamine 3000 (Invitrogen). For the construction of dual-luciferase reporter vectors, the sequences with the wide type or mutant type binding sites of circ_0062020 in miR-615-5p were respectively inserted into pMIR-Report vectors (Promega, Madison, WI, USA) and named as WT-circ_0062020 and MUT-circ_0062020. Similarly, WT-TRIP13-3ʹuntranslated region (WT-TRIP13-3ʹUTR) and MUT-TRIP13-3ʹUTR represents the pMIR-Report vectors (Promega) contained the sequences with wild type or mutant type binding sites of miR-615-5p in TRIP13 3ʹUTR. Finally, the luciferase activity was tested by a Dual-Luciferase® Reporter Assay System (Promega). Renilla luciferase activity served as an internal normalization of firefly activity values.
Pmir report vector
Manufactured by Promega
Sourced in United States, United Kingdom
The PMIR-REPORT vector is a plasmid designed for the expression and detection of reporter genes in mammalian cells. The vector contains a multiple cloning site for the insertion of your gene of interest, as well as a reporter gene for monitoring expression. The reporter gene encodes a protein that can be easily detected, allowing for the quantification of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (35 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (15 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (5 mentions)
Prl tk vector, by Promega (5 mentions)
Dual luciferase assay kit, by Promega (4 mentions)
Dual glo luciferase assay system, by Promega (3 mentions)
Dual luciferase assay system, by Promega (3 mentions)
Prl tk, by Promega (2 mentions)
Pmir report mirna expression reporter vector, by Thermo Fisher Scientific (2 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (2 mentions)
X tremegene transfection reagent, by Roche (2 mentions)
Genetailor site directed mutagenesis system, by Thermo Fisher Scientific (2 mentions)
Prl tk renilla luciferase control vector, by Promega (2 mentions)
Pmir report firefly luciferase mirna expression reporter vector, by Thermo Fisher Scientific (2 mentions)
Pmir report vector, by Thermo Fisher Scientific (2 mentions)
M 280 streptavidin magnetic beads, by Merck Group (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
Dual luciferase reporter gene assay system, by Promega (1 mentions)
Mirna nc, by RiboBio (1 mentions)
71 protocols using pmir report vector
1
Dual-Luciferase Reporter Assay for circRNA-miRNA Interaction
2
Dual Luciferase Assay for 3'UTR Regulation
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For dual luciferase report assay, the Reporter constructions were performed by synthesizing wild type or mutated 3'UTR of PBX1 and ACAN and subcloned into pMIR-REPORT vector (Promega, WA, USA), which all these procedures were done by GeneChem Corp (Shanghai, China). Before the experiments, the HEK293T cells were seeded in 96-well plates for 24 hours, and a mixture of the pMIR-REPORT vector (wild type or site mutated plasmid) and miRNAs mimics or scramble control were co-transfected into cells, and a PRL-TK vector (carrying Renilla luciferase) was also co-transfected and served as internal control (Promega, Madison, USA). The transfection uses Lipofectamine 2000 (Invitrogen) with 50 ng of pMIR-REPORT vector (carrying firefly luciferase), 25ng of PRL-TK vector (carrying Renilla luciferase) and 10nmol of miRNA mimics or scramble controls. After transfection for 48 hours, the Dual-Luciferase Reporter Assay System (Promega, Madison, USA) was used to detect the luciferase activity. And Light intensity was normalized by Firefly luciferase.
3
Construct Luciferase Reporter Vectors
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To construct luciferase reporter vectors circ_0084615-wt or ONECUT2 3’UTR-wt, the sequences of circ_0084615 or ONECUT2 3’UTR including miR-599 binding sites (GACACAA) were introduced into pmiR-REPORT™ vectors (Promega, Madison, WI, USA). Circ_0084615-wt or ONECUT2 3’UTR-wt was constructed by mutating the binding sites. Then the vectors were transfected into SW620 and DLD-1 cells in combination with miR-599 or miR-NC, followed by measurement of the luciferase intensity.
4
Luciferase Reporter Assay for miRNA Targets
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The fragments of circ-PITX1 or CCND2 3ʹUTR including the putative binding sites or mutant sites of miR-1248 were cloned into the pMIR-REPORT vectors (Promega, Madison, WI, USA), generating the wild-type (WT) or mutate-type (MUT) vectors, respectively. The vectors were co-transfected with miR-1248 mimic or miR-NC into NSCLC cells and incubated for 48 h. Luciferase activity was assessed by Dual-Luciferase Reporter Assay System (Promega).
5
Validating miR-181a-5p Targeting of NDRG2
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Targeted 3ʹ-untranslated regions (UTR) of NDRG2 (wild-type; wt NDRG25) and its corresponding mutant (mt) NDRG2 fragments were inserted into pMIR-REPORT vectors (Promega, Madison, WI, USA), respectively. HEK293T cells were seeded into 24-well plates for 48 h before the transfection. Then the cells were co-transfected using Lipofectamine 2000 (Invitrogen) with wt NDRG25 or mt NDRG2 and miR-181a-5p or miR-ctrl. After 48 h, luciferase activity was detected by Steady-Glo Luciferase Assay System (Promega), which was quantified by the firefly/Renilla luciferase activity ratio.
On the other hand, the targeted relationship was also verified using the RNA pull-down assay. Briefly, biotinylated wt-miR-181a-5p and mut-miR-181a-5p were transfected into cells for 48 h. The cells were lysed followed by incubated with M-280 streptavidin magnetic beads (Sigma) at 4°C for 3 h. After washing with lysis buffer, low salt buffer, and high salt buffer, The expression of NDRG2 was assessed by RT-qPCR.
On the other hand, the targeted relationship was also verified using the RNA pull-down assay. Briefly, biotinylated wt-miR-181a-5p and mut-miR-181a-5p were transfected into cells for 48 h. The cells were lysed followed by incubated with M-280 streptavidin magnetic beads (Sigma) at 4°C for 3 h. After washing with lysis buffer, low salt buffer, and high salt buffer, The expression of NDRG2 was assessed by RT-qPCR.
6
Validating miR-20b Regulation of NFAT5 and CAMTA1
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3′-UTR luciferase reporter assays were performed in human HEK293T cells. The 3′-UTR of NFAT5 and CAMTA1 with potential miR-20b binding sites was cloned from the genome of Jurkat cells and then placed into pmiR-REPORT vectors (Promega, Madison, WI, USA). HEK293T cells were cotransfected with the constructed luciferase reporter plasmids and miRNA. Renilla luciferase plasmids were cotransfected and used for normalization of transfection efficiency. After 48 h of transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega).
7
Investigating miR-140-3p Binding to circ-PREX1 and WNT5B
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Mutant types of circ-PREX1 (MUT-circ-PREX1) and MUT-WNT5B 3′UTR were acquired from Yeasen Biotech, thus the mutants showed no miR-140-3p binding sites. The wild types of circ-PREX1 (WT-circ-PREX1) and WT-WNT5B 3′UTR were separately inserted in pmiR-REPORT™ vectors (Promega, Madison, WI, USA), as well as the mutants. C28/I2 cells were transferred in 24-well plate and co-transfected with pmiR vectors (400 ng) and mimics (40 pmol) of miR-140-3p or miR-NC. After co-transfection for 48 h, luciferase activities were detected using Dual-Glo Luciferase Assay System (Promega).
8
Luciferase Assay for miR-138-5p Targets
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The fragment of circ-TLK1 or SOX4 mRNA containing the putative binding sites or mutant sites of miR-138-5p was cloned into the pMIR-REPORT vectors (Promega, Madison, WI, USA). Cells were cultured in 24-well plates in advance and cotransfected with the reporter vectors and miR-138-5p mimics or NC mimics using Lipofectamine 2000. At 48 h posttransfection, the cells were lysed, and their luciferase activities were measured by Dual-Luciferase Reporter Assay System (Promega).
9
Luciferase Assay for Circular RNA-miRNA Interactions
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Wild type (WT) and mutant (MUT) fragments of circ_0000005 sequences were inserted into pMIR-REPORT vectors (Promega, Madison, WI, USA). Similarly, WT and MUT fragments of Tspan3 3'-UTR with the binding sites for miR-139-5p were cloned into pMIR-REPORT vectors. After the reporter vectors were constructed, the cells were co-transfected with these reporter vectors and miR-139-5p mimics or miR-NC.
48 h later, dual-luciferase reporter gene assay system (Promega, Madison, WI, USA) was employed to detect the luciferase activity in the cell lysate of each group.
48 h later, dual-luciferase reporter gene assay system (Promega, Madison, WI, USA) was employed to detect the luciferase activity in the cell lysate of each group.
10
Dual Luciferase Assay for miRNA Targeting
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For dual luciferase assays, luciferase reporter pMIR-REPORT vectors (Promega, USA) containing the 3′ UTR of BCL2L11 with its wild-binding site (WT) or with mutant-binding sites (MUT) were specifically synthesized (GenePharma, China). HEK-293T cells were transfected with 10 ng of each pMIR-REPORT construct along with miRNA mimics/inhibitors or scrambled/negative controls and Lipofectamine 2000 reagent (Invitrogen, USA). After 48 h, the cells were lysed, and the firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega, USA). Each fragment containing a putative miRNA-binding site was cloned into the pMIR-REPORT vector immediately downstream of the Renilla luciferase gene. The results are presented as the ratio of Renilla luciferase activity to firefly luciferase activity.
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