The FOXF2 3′-UTR fragment containing the predicted potential has-miR-19a-3p binding site was amplified using polymerase chain reaction (PCR) and subcloned downstream of the luciferase gene of the pMIR-reporter luciferase vector (Promega, USA). Human embryonic kidney 293 T cells were cultured in a 96-well plate and transfected with Lipofectamine 2000 (Thermo Fisher Scientific). Luciferase activity was measured using a dual-luciferase reporter assay system (Promega).
Pmir reporter luciferase vector
Manufactured by Promega
Sourced in United States
The PMIR-reporter luciferase vector is a plasmid-based system designed for the expression and measurement of microRNA (miRNA) activity. The vector contains a luciferase reporter gene that is regulated by a miRNA-responsive promoter, allowing for the quantification of miRNA levels in a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pmir report luciferase vector, by Thermo Fisher Scientific (1 mentions)
Veritas microplate luminometer, by Promega (1 mentions)
Renilla luciferase control vector, by Promega (1 mentions)
4 protocols using pmir reporter luciferase vector
1
Luciferase Assay for miRNA Binding
2
Validation of miR-320 Binding to ELF3 3'UTR
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Bioinformatics tools (TargetScanHuman 7.1, http://www.targetscan.org/vert_71/ ) were used to predict the miR-320 binding sites of ELF3. The recombinant pMIR-reporter luciferase vector was applied for luciferase assays. The wild or mutant type of ELF3-3′ untranslated region (3′UTR; containing miR-320 binding sites) were synthesized and then cloned into the pMIR-reporter luciferase vector (Promega Corporation). MCF-7 cells were co-transfected with miR-320 mimic and vector using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). MCF-7 cells were seeded into 48-well plates at a density of 5×104 cells per well. After transfection for 48 h at 37°C, the Dual Luciferase Reporter assay system (Promega Corporation) was used to measure the luciferase activity. The relative luciferase activity was normalized to Renilla luciferase activity 48 h after transfection.
3
Luciferase Assay for miR-150 Binding Targets
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The fragment 5′-aaaTGGAAAGATTAATTGGGAGtgg-3′ (position 1839) from Malat1 and the fragment 5′-tcaagcaatcgagatTTGGGAGc-3′ (position 52) from eIF4E 3′-UTR containing the predicted miR-150 target sites were chemically synthesized and individually inserted into the pMIR-luciferase reporter vector (Promega, Madison, WI, USA), and finally named as pMIR-Malat1-wt and pMIR-eIF4E-wt, respectively. The corresponding mutants were created by mutating the miR-150 seed region binding site (seed sequence binding fragment 5′-TGGAAAGATTAATTGGGAG-3′ from Malat1 changed to 5′-CCCTGGCTCCGGCCACCTC-3′, and seed sequence binding fragment 5′-TTGGGAG-3′ from eIF4E 3′-UTR changed to 5′-ACCCATA-3′), which were named as pMIR-Malat1-mut and pMIR-eIF4E-mut, respectively. ASMCs were cotransfected with pMIR-luciferase reporter vector containing Malat1 fragment or eIF4E 3′-UTR with wide type or mutant binding sites with miR-150, and mimic-NC or miR-150 mimic by using Lipofectamine 2000. Cells were harvested 48 h after transfection, and the luciferase activities were determined by using the Dual Luciferase Reporter Assay System (Promega).
4
Validating miRNA Target Interactions
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Human genomic DNA was used to amplify the 3′ UTR of miRNA target genes by PCR and the amplified PCR fragment was cloned into the pMIR-REPORT Luciferase Vector (Part Number AM5795, Life Technologies) between SpecI and SacI restriction sites. For the EGFR-3′ UTR reporter, primers 5′-GACTACTAGTCTTCAATGGGCTCT TC CAACAAGG-3′ and 5′-GACTGAGCTCGGTCCAAA TGCTGATGAATCC-3′ were used to amplify a fragment of 532 bp containing two predicted miR-7 seed binding sequences. For the RAF1–3′ UTR reporter, primers 5′-GACTACTAGTGAAGTAAGGTAGCAGGCAGTCC-3′ and 5′-GACTGAGCTCTGAGGGACCATCAGATAAC TG-3′ were used to amplify a fragment of 555 bp also containing two seed binding miR-7 target sequences. ACC cells were co-transfected with the pMIR Luciferase Reporter Vector plus Renilla Luciferase Control Vector (Promega) along with the miRNA using Lipofectamine 2000 (Life Technologies) and the luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega) using a luminometer (Veritas Microplate Luminometer, Turner Biosystems, CA, USA). Relative luciferase activity was quantified by calculating the firefly to Renilla luciferase signal ratio.
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