PDE7B 3’-UTR luciferase reporter gene plasmid was constructed by inserting the entire human PDE7B 3’-UTR sequence into the pMiR vector (Invitrogen). PDE7B 3’-UTR containing mutation at miR-200c targeting site was generated through site-directed mutagenesis on wild-type plasmid using QuickChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). To determine the effect of PDE7B 3’-UTR on luciferase activity, cells were transfected with empty pMiR or pMiR containing wild-type or mutant PDE7B 3’UTR for 2 days and then lysed for measurement of luciferase activity. The trransfection efficiency was normalized by including pTK-RLuc plasmid (1:200 ratio) in all transfection experiments and Renella luciferase activity was determined for standardization. Dual-Luciferase® Reporter Assay System (Promega, Madison, WI) was used to measure both firefly and Renella luciferase activities.
Pmir vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR vectors are a set of plasmid-based expression vectors designed for microRNA (miRNA) overexpression and knockdown studies in mammalian cell lines. These vectors provide a platform for the cloning and expression of miRNA precursors or miRNA inhibitors, enabling researchers to manipulate miRNA levels in their experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Pre mir 30a, by Merck Group (2 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Anti p53 do 1, by Santa Cruz Biotechnology (1 mentions)
Anti p21, by Thermo Fisher Scientific (1 mentions)
Anti bim, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000 transfection regent, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay, by Beyotime (1 mentions)
293 cells, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
14 protocols using pmir vector
1
PDE7B 3'-UTR Luciferase Reporter Assay
2
PDE7B 3'-UTR Luciferase Reporter Assay
3
Luciferase Assay for miR-22 Targeting Sirt1 3'-UTR
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The pMIR vector (Ambion, Austin, TX, USA) was used to express luciferase and as the template vector for 3′‐UTR reporter assays. Wild‐type and mutant 3′‐UTRs from Sirt1 mRNA were cloned and inserted into the 3′‐UTR of pMIR. Then, L02 cells were transfected with pMIR and miR‐22 mimic using Lipofectamine 2000, and pRL‐CMV was used as an internal reference. The cells were lysed for the analysis of relative luciferase activity after 36 h post‐transfection. The activities of firefly and Renilla luciferases were measured using the dual‐luciferase reporter assay system kit (Promega, Madison, WI, USA) according to the instructions. The relative luciferase activities were calculated as firefly luciferase (pMIR) divided by Renilla luciferase (pRL‐CMV).
4
Validation of miR-30a Binding Sites
5
Validation of miR-30a Binding Sites
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Three PCR-generated fragments containing the miR-30a binding sites (one in NOTCH1, two in NOTCH2) (Supplementary Figure 1 ) were individually cloned in the 3′UTR of the luciferease gene (pMIR vector, Ambion). Site directed mutagenesis was used to modify three of the six “seed sequence” nucleotides in each construct, as we described23 (link). After sequencing verification, these constructs were cotransfected in HEK-293 cells with the pCMVβ-gal plasmid (5ng) and 100 nM each of chemically synthesized pre–miR-30a or pre–miR negative control oligonucleotides (Sigma). Cells were harvested 48 h after transfection, and luciferase and β-galactosidase activities measured, as we described24 (link). The primers sequences are listed in Supplementary Table 3 .
6
Generation and Validation of miR-139 Reporters
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The pGL3-miR-139 luciferase reporter plasmid was constructed from the miR-139 promoter with primers as listed in Supplementary file 1 . The fragment was inserted into pGL3 at the MluI and XhoI sites. The pGL3-miR-139-mut was generated by site-directed mutagenesis with primers listed in Supplementary file 1 using pGL3-miR-139 as template. The pMIR-PDE4D and pMIR-PDE4D mutant plasmids were constructed by inserting the miR-139-5p-targeted PDE4D mRNA-coding sequence or its mutant into the pMIR vector (Ambion) at the SpeI and HindIII sites. The pSIF-H1-miR-139-5p was constructed by inserting annealed oligos as listed in Supplementary file 1 into the pSIF-H1 vector at BamHI and EcoRI sites, as per manufacturer’s instruction. The anti-p21 (Thermo Fisher Scientific, Waltham, MA, RRID: AB_10986834 ), anti-p53 (DO-1, Santa Cruz, Dallas, TX, RRID: AB_628082 ), anti-PDE4D (Aviva Systems Biology, San Diego, CA, RRID: AB_10879817 ), and anti-BIM (Cell Signaling, Danvers, MA, RRID: AB_10692515 ) antibodies used here were commercially purchased. The anti-MDM2 (2A10 and 4B11) antibodies were described previously (Jin et al., 2002 (link)).
7
Validation of miR-214-3p and CDIP1 Interaction
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The relationship between miR-214-3p and CDIP1 was predicted using TargetScan (https://www.targetscan.org/vert_80/ ). The CDIP1 3’-UTR, which includes the miR-214-3p binding site or a mutated target site, was amplified and fixed onto pMIR vectors (Ambion, USA) to obtain wild-type CDIP1 plasmid (CDIP1-WT) or CDIP1 mutated plasmid (CDIP1-MUT). The CDIP1-WT or CDIP1-MUT and miR-214-3p mimics or mimic control were co-transfected into 293T cell by Lipofectamine 2000 and cultivated for 48 h following the instructions. Dual-Luciferase Reporter Assay System (cat. no. E1910; Promega) was applied to check the luciferase activity [25 (link)].
8
Investigating miR-192-5p Regulation of lncRNA WAC-AS1
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The data base known as starBase was used to identify the relationship between miR-192-5p and lncRNA WAC-AS1. The 3’UTR of lncRNA WAC-AS1, containing the miR-192-5p binding site or mutated target site, was synthesized by carrying out a genomic PCR (ELK Biotechnology) and was cloned into pMIR vectors (Ambion, Austin, TX, USA) to construct the reporter vector lncRNA WAC-AS1 wild-type (lncRNA WAC-AS1-WT) or lncRNA WAC-AS1 mutant-type vector (lncRNA WAC-AS1- MUT). Next, 293T cells were transfected with lncRNA WACAS1- WT or lncRNA WAC-AS1-MUT combined with miR-192-5p mimic or mimic control using Lipofectamine 2000 transfection regent (Invitrogen). The transfected cells were incubated for 48 h and dual-luciferase reporter assay (Beyotime Technology, Jiangsu, China) was performed. Firefly luciferase activity of the plasmids was normalized to Renilla luciferase activity.
9
miR-421 Regulation of BMF Expression
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TargetScan was used to predict the potential targets of miR-421 [18 ]. The 3’-UTR of BMF containing miR-421 binding sites was cloned into pMIR vectors (Ambion, USA) to generate the BMF wild-type (BMF-WT) or BMF mutated (BMF-MUT) plasmid. For the reporter activity analysis, 293 T cells were cotransfected with BMF-WT or BMF-MUT plasmids and miR-421 mimic or mimic control using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. After 24 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA) [19 (link)].
10
Cx43 Regulation by miR-206 Binding
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TargetScan bioinformatics software (http://www.targetscan.org/vert_72/ ) was used to predict the binding sites between miR-206 and Cx43. The 3'-untranslated region of Cx43, which contains the miR-206 binding site or mutated target site, were synthesized using genomic PCR and cloned into pMIR vectors (Ambion; Thermo Fisher Scientific, Inc.) to construct the reporter vector Cx43 wild-type (Cx43-WT) or Cx43 mutated-type (Cx43-MUT). The 293 cells (American Type Culture Collection) were transfected with Cx43 WT or MUT combined with miR-206 mimics or mimics control using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated for 48 h, according to the manufacturer's protocol. A Dual-Luciferase Reporter Assay system (Promega Corporation) was used to assess the luciferase activity, and the data were normalized to Renilla luciferase activity.
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