TargetScan Human 5.1 software (22 (link)) (www.targetscan.org ) was used to determine the putative target of miR-423-3p. The wild-type (WT) PANX2 3′-untranslated region (UTR) was constructed by PCR, which was performed by Yearthbio (Changsha, China), and inserted into the pMIR-REPORT miRNA Expression Reporter vector (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The mutant type (MT) of PANX2 3′-UTR was constructed using the Easy Mutagenesis System kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol, and then inserted into the pMIR-REPORT miRNA Expression Reporter vector (Thermo Fisher Scientific, Inc.). U251 and U87MG Uppsala cells were co-transfected with WT PANX2-3′UTR plasmid or MT PANX2-3′UTR plasmid, and miR-NC or miR-423-3p mimics, using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.). Following transfection at 37°C for 48 h, the activity of Renilla luciferase and firefly luciferase were determined using a dual-luciferase reporter assay system (Promega Corporation), 48 h after transfection. The activity of Renilla luciferase was normalized to that of the firefly luciferase.
Pmir report mirna expression reporter vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The PMIR-REPORT miRNA expression reporter vector is a tool designed for the detection and measurement of microRNA (miRNA) expression levels. It functions by incorporating a miRNA target sequence that allows for the monitoring of miRNA activity through a reporter gene output.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (23 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (10 mentions)
Dual luciferase assay system, by Promega (8 mentions)
Prl tk renilla luciferase vector, by Promega (5 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (3 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (3 mentions)
Easy mutagenesis system kit, by Promega (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Site directed mutagenesis kit, by Takara Bio (3 mentions)
Neolite reporter gene assay system, by PerkinElmer (2 mentions)
Turbofect reagent, by Thermo Fisher Scientific (2 mentions)
Pmir report vector, by Promega (2 mentions)
Prl sv40 vector, by Promega (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Pcdh cmv ef1 gfp puro, by System Biosciences (2 mentions)
Mir 101 mimic, by GenePharma (2 mentions)
Prl tk, by Promega (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (1 mentions)
62 protocols using pmir report mirna expression reporter vector
1
Determining miR-423-3p Targets Using Luciferase Assay
2
Regulation of GRB2 by miR-329
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The 3′-UTR of GRB2 was amplified by PCR using cDNA from SW1990 cells and cloned into Hind III-MluI sites of the pMIR-REPORT miRNA expression reporter vector (Applied Biosystems). The mutant GRB2 3′-UTR was constructed to mutate three intermittent sites complementary to the miR-329 seed-region. The miR-329 mimics or control and luciferase reporter vector were co-transfected into SW1990 cells. 48h after transfection luciferase activities were measured with the dual-luciferase assay kit (Promega) according to manufacturer's instructions.
3
Characterization of miR-218 Regulation of CDCP1
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The plasmid containing sequences of primary miR-218 was constructed as previously described16 (link). The CDCP1 3′-UTR was amplified from leukocyte genomic DNA and inserted into the pMIR-REPORT miRNA Expression Reporter Vector (Applied Biosystems, Carlsbad, CA, USA) by adding the SpeI and MluI sites. Three miR-218 binding sites were predicted in the CDCP1 3′-UTR using miRSystem19 (link), located at 1,840–1,860 bp, 2,100–2,119 bp, and 2,951–2,971 bp relative to the transcription start site. Mutations of the miR-218 binding sites in the CDCP1 3′-UTR or mutations of the primary miR-218 sequence were made using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. These reporter plasmids containing the CDCP1 3′-UTR, primary miRNA, and Renilla luciferase sequences were co-transfected into the indicated cells as previously described. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Lung cancer cells expressing doxycycline-inducible pri-mir-218 were generated by infecting Bm7brmx2 cells with a lentiviral tet-on-pri-mir-218 plasmid to generate stable cell lines, as previously described16 (link).
4
Cloning and Mutating VEGFA 3'-UTR
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We amplified a 343-bp VEGFA 3'-UTR from SGC-7901 cells cDNA using the following PCR primers: VEGFA3-UTR-5, 5'-ATCGGTGACAGTCACTAG-3' and VEGFA3-UTR-3, 5'-TACGGATAAACAGTAGCA-3'. The amplified fragment was first cloned into pCR2.1-TOPO vector (Invitrogen, USA) and subsequently cloned into the SpeI and HindIII sites of the pMIR-REPORT miRNA expression reporter vector (Applied Biosystems, USA). The first six nucleotides complementary to the miR-29a seed-region were mutated from the mutant constructs using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, USA).
5
Characterization of RANK 3'-UTR Regulation
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The Tnfrsf11a (RANK) 3’-UTR was amplified by PCR from mice-isolated genomic DNA. The Tnfrsf11a (RANK) 3’-UTR was amplified by PCR from mouse OCP-isolated genomic DNA. The pMIR-RANK-3’-UTR construct was digested with HindIII and SacI, and the generated fragment was inserted into the corresponding restriction sites of the pMIR-REPORT miRNA Expression Reporter vector (Applied Biosystems, Carlsbad, CA). Two miR-218–2-3p binding sites in the Tnfrsf11a 3’-UTR were predicted using the miRanda-mirSVR application system and were located at 3,365–3,370 bp and 3,458–3,467 bp relative to the transcription start site. Mutations were made in the miR-218–2-3p binding sites using primers described in Fig. 3 G in the Tnfrsf11a 3’-UTR using the Muta-DirectTM Site-directed mutagenesis kit (Intron, Seoul, Korea) in accordance with the manufacturer’s protocol.
6
CDH2 3'UTR Cloning and Mutation
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The CDH2 3′-UTR was amplified by PCR from genomic DNA isolated from human blood. The pMIR-CDH2-3′UTR construct was digested with SpeI and MluI, and the generated fragment was inserted into the SpeI-MluI sites of the pMIR-REPORT miRNA Expression Reporter Vector (Applied Biosystems, Carlsbad, CA, USA). Three miR-218 binding sites in the CDH2 3′-UTR were predicted using miRSystem [29] (link), and these sites were located at 2,671–2,691 bp, 2,740–2,760 bp, and 3,571–3,591 bp relative to the transcription start site. Mutations were made in the miR-218 binding sites in the CDH2 3′-UTR using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocol.
7
Generating Reporter Constructs for miR-1183
8
STAT3 3'UTR Luciferase Assay
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The putative binding sites of the 3′-untranslated region (UTR) of the human genes for miR-340-5p targeting were predicted using the TargetScan Human computational methods (http://www.targetscan.org/ ). The partial 3′-UTR fragment of STAT3, the wild STAT3 including predicted binding sites, and the mutant STAT3 inserts with an opposite mutation in the miRNA seed sequence binding sites, were cloned into the pMIR-REPORT™ miRNA Expression Reporter Vector (Applied Biosystems). All primers were designed as shown in Table 1 . HEK293T cells were seeded in triplicate at a density of 2 × 105 cells per 24-well plate. Luciferase activity was monitored using the Luciferase Assay Kit (Promega) and normalized to Renilla luciferase activity, as previously described [21 (link)].
9
Cloning and Mutating CD44 3'UTR
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To clone fragments of the potential downstream target genes of miR-204, a PCR-based method was used. Specific primers were designed using a bioinformatics search in GenBank. Total RNA was extracted from the H1975 cell line, and the cDNA library was obtained. We amplified the 3′ UTR of the CD44 gene sequence from the cDNA library and cloned it into the pMIR-REPORT miRNA expression reporter vector (Applied Biosystems, Foster City, CA). A mutant 3′ UTR of CD44 harboring a mutant sequence of the miR-204 binding site was constructed by the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The cells were cultured in 24-well plates at a density of 3 × 104 cells per well for 24 h before transfection. Subsequently, cells were transfected with a Luc-putative gene vector or Luc-putative gene mutation vector. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega) after 24 h of incubation.
10
Dual-Luciferase Assay for miR-29a Targeting PI3Kp85α
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The wild type pMIR-PI3Kp85α luciferase plasmid was constructed by cloning mouse PI3Kp85α-3′UTR sequence into the pMIR-REPORT™ miRNA Expression Reporter Vector (Applied Biosystems, Foster City, CA, USA), while the pMIR-PI3Kp85α-Mut luciferase plasmid was substituted with five mismatched sites (Figure 4 ). The plasmids were purified using EasyPrep EndoFree Maxi Plasmid Extraction Kit (BIOTOOLS, Ltd., New Taipei, Taiwan). 9 × 105 HepG2 cells were seeded at a 6 cm culture dish. After 24 h, 3 µg of pMIR-PI3Kp85α luciferase plasmid or pMIR-PI3Kp85α-Mut plasmid was introduced by TurboFect reagent (Thermo Fisher Scientific, Rockford, IL, USA). After another 24 h, 25 nM of miR-29a precursor (mimic-miR-29a, GE Healthcare Dharmacon, IN, USA) or miR control sequence (GE Healthcare Dharmacon) were introduced by using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) as per the manufacturer’s standard protocol. After incubated for 24 h at 37 °C, the cells were lysed for the detection of luciferase signal with Neolite Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA).
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