The artificially synthesized PlGF 3' untranslated region (UTR) gene fragment was constructed into a pMIR reporter (Promega, Madison, WI, USA). A complementary sequence with mutation (MUT) of the seed sequence was designed based on the wild type (WT) of PlGF and constructed into the pMIR-reporter reporter plasmid. The correctly sequenced luciferase reporter plasmids WT and MUT were co-transfected with miR-214 mimic and miR-214 NC into 293T cells (Hunan Fenghui Biological Co., Ltd., Hunan, China) at the second passage. Forty-eight hours after transfection, the cells were collected and lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
Pmir reporter
Manufactured by Promega
Sourced in United States
The PMIR-reporter is a lab equipment product designed for nucleic acid detection and quantification. It provides a sensitive and reliable method for measuring mRNA or miRNA levels in biological samples. The PMIR-reporter utilizes a proprietary technology to enable accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (9 mentions)
Fluorescence detector, by Promega (2 mentions)
Dual luciferase reporter assay system kit, by Promega (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Dual luciferase assay, by Promega (1 mentions)
Dual luciferase reporter gene assay system, by Promega (1 mentions)
Kapa hifi dna polymerase, by Roche (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Nf κb luciferase reporter, by Thermo Fisher Scientific (1 mentions)
Luciferase activity detection kit, by Promega (1 mentions)
Site direct mutagenesis kit, by Takara Bio (1 mentions)
Dual luciferase activity system, by Promega (1 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Stop glo reagent, by Promega (1 mentions)
25 protocols using pmir reporter
1
Luciferase Assay for miR-214 Targeting
2
Circular RNA Regulates AURKA Expression
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The bioinformatics website predicted that circ_0061265 could bind with miR-885-3p. Artificially synthesized circ_0061265 or AURKA 3’UTR gene fragments were introduced into pMIR-reporter (Promega, Madison, WI, USA). Subsequently, the complementary sequence mutation site of the seed sequence was designed on the basis of wild type (WT) AURKA or circ_0061265 and then constructed into the pMIR-reporter plasmid. The correctly sequenced WT and mutant type (MUT) luciferase reporter plasmids were co-transfected with NC mimic and miR-885-3p mimic into NCI-N87 cells (Bnbiotech, Shanghai, China) for 48 h. Thereafter, the luciferase activity was detected by means of Dual-Luciferase Reporter Assay System (Promega).
3
TRPM6 Regulation by miR-202-3p
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According to the prediction on the http://www.targetscan.org/vert72/website , TRPM6 is the target of miR-202-3p. The fragment of a synthetic TRPM6 3ʹUTR gene was inserted into pMIR-reporter (Promega, Madison, WI, USA) using endonuclease sites SpeI and Hind III. The complementary sequence mutation (MUT) sites of seed sequences were designed according to the wild type (WT) of TRPM6. The target fragments were inserted into the pMIR-reporter plasmid using restriction enzyme digest and T4 DNA Ligase. HEK-293T cells (Shanghai BeinuoBio Biological Technology Co., Ltd., Shanghai, China) were transfected with miR-202-3p and luciferase reporter plasmids WT and MUT, respectively. After transfection for 48 h, the supernatant was discarded and then cells were washed with phosphate buffered saline (PBS). Cells were then lysed for 5–10 min at room temperature with diluted cell lysis, and then triturated. The cell fragments were removed by centrifugation at 1610 × g for 5 min. A total of 50 μL luciferase was added into each group, and transferred to the detection plate. Luciferase activity was confirmed by the dual-channel fluorescence of luciferase assay kit.
4
Prediction and Validation of miR-497 Binding Sites in IRS1
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Prediction of miR-497 binding sites was performed using TargetScan software using the key words ‘IRS1’ and ‘human species’. TargetScan (www.targetscan.org ) and miRanda (www.microrna.org ) predict biological targets of miRNAs by searching for the presence of conserved 8mer, 7mer and 6mer sites that match the seed region of each miRNA (27 (link)). A fragment of 3′-UTR of IRS1 containing the putative miR-497 binding site was amplified by PCR. To generate a construct containing the mutant miR-497 binding site, two nucleotides corresponding to the 5′-seeding region of the miR-497 binding site on the wild type fragment were substituted. Its complementary sequence in the 3′-UTR of IRS1 (UGCUGCU) was replaced by UCCACCA. The PCR products were digested using SacI and HindIII, inserted into pMIR-REPORTER (Promega Corporation, Madison, WI, USA) and validated by DNA sequencing. Constructs were transfected into HEK-293 cells in 24-well plates and co-transfected with miR-497 or miR-NC. Luciferase assays were performed 24 h post-transfection using the Dual Luciferase Reporter Assay system (Promega Corporation).
5
Validating miR-374 Regulation of Gadd45a
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The biological prediction website, www.microRNA.org , identified that Gadd45a was a direct target of miR-374. The synthetic Gadd45a 3′-UTR gene segment was introduced into pMIR-reporter (Promega, Madison, WI, U.S.A.) using restriction enzymes, SpeI and HindIII. The mutation site in complementary sequences of wild-type (WT) Gadd45a was designed. After restriction endonuclease digestion, the target segments were inserted into the pMIR-reporter plasmids using the T4 DNA ligase. The identified luciferase reporter plasmids WT and mutant-type (MUT) were respectively co-transfected into HEK-293T cells (Shanghai North Connaught Biotechnology Co. Ltd., Shanghai, China) with miR-374. After 48 h, the cells were collected and lysed, and luciferase activity was detected using a luciferase assay kit.
6
Luciferase Reporter Assay for miRNA Target Validation
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The reverse complementary sequence of miR-133b was inserted into pMIR-reporter (Promega, WI, USA) to generate a reporter system (pMIR-133b) to detect mature miRNA expression in 293 T cells. The 3′ UTR of the human FSCN1 was PCR amplified and cloned into pMIR-reporter downstream of the firefly luciferase gene to generate the corresponding reporters. Mutations at the miRNA binding site in these mRNA sequences were created using bridging PCR. For miRNA targets analysis, the 293 T cells were co-transfected with 0.4 μg of the reporter construct, 0.02 μg of pRL-TK vector, and 5 pmol of miRNA mimic or scramble controls. Cells were harvested 48 h post-transfection and assayed with Dual Luciferase Assay (Promega, WI, USA) according to manufacturer’s instructions. All transfection assays were carried out in triplicates.
7
Regulation of Rab10 by miR-519d
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The MicroRNA.org website was used to analyze the target genes of miR-519d. The dual-luciferase reporter gene assay was used to determine whether Rab10 gene was a direct target of miR-519d. The synthetic Rab10-3ʹUTR gene fragment was introduced into a pMIR-reporter (Promega, Madison, WI, USA) using the endonucleases SpeI and HindIII. The complementary sequence mutation sites of the seed sequence were designed on the Rab10 wild-type (WT). After restriction endonuclease digestion and ligation with T4 DNA ligase, the target fragments were inserted into the pMIR-reporter reporter plasmid. The correctly sequenced luciferase-reporter plasmid WT or mutation (MUT) were respectively co-transfected with miR-519d and negative control (NC) into HEK-293T cells (Beinuo Life Science, Shanghai, China). After being transfected for 48 h, the cells were collected and lysed. The luciferase activity was detected by the firefly luciferase assay kit.
8
Validating RHPN2 as miR-205 Target
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The biological prediction website microRNA.org was used to verify whether RHPN2 was a target gene of miR-205, which was further verified by dual-luciferase reporter gene essay. The artificially synthesized RHPN2 3’untranslated region (3’UTR) gene fragment was introduced into a pMIR-reporter (Promega Corporation, Madison, WI, USA) through the endonuclease sites SpeI and Hind III. The complementary sequence mutation sites of seed sequences were designed on wild type (WT) of RHPN2 3’UTR. After restriction endonuclease digestion, the target fragment was inserted into the pMIR-reporter reporter plasmids via T4 DNA ligase. The WT and mutant (MUT) plasmids were co-transfected with miR-205 mimic and mimic NC into HEK-293 T cells (Beinuo Life Science, Shanghai, China). After transfection for 24 h, the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) was employed to detect luciferase activity. The experiment was repeated three times independently.
9
Validating the miR-199a-3p-3p Binding to PIK3CA
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The website for biological prediction (www.microRNA.org ) was used to predict the binding region between PIK3CA and miR-199a-3p-3p. First, we constructed the ABCF2 30UTR gene fragment and inserted it into the pMIR-reporter (Promega, Madison, Wisconsin, USA). Next, a mutant (MUT)-binding site was designed using a complementary sequence of wild-type (WT) PIK3CA seeds. Next, the pMIR-reporter plasmid was constructed. HEK-293 T cells were co-transfected with PIK3CA-MUT or PIK3CA-WT, the luciferase reporter gene plasmid with correct sequences, with miR-199a-3p-3p mock or mock negative controls (NC) (Beinuo Life Science Co., Ltd., Shanghai, China) for 48 h. After cell collection and lysis, the luciferase activity was detected using the dual-luciferase reporter gene assay system (Promega).
10
CircRNA overexpression plasmid construction
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CircRNA overexpression plasmid was constructed by cloning the RTN4 exon 4 and 5 into pcDNA3.1 (+) circRNA mini vector, which was a gift from Jeremy Wilusz (Addgene plasmid # 60648) [9 , 11 (link)]. pmiR-circRTN4 reporter plasmid for luciferase assay was constructed by cloning circRTN4 sequence into region directly downstream of the firefly luciferase gene in the pmiR-Reporter (Promega, Madison, WI, USA). Mutation in the miRNA binding site of the pmiR-circRTN4 reporter plasmid and the RAB11FIP1 binding site of the pcDNA3.1 (+)-circRTN4 plasmid were generated using KAPA HiFi DNA Polymerase (KapaBiosystem, St. Louis, MO, USA) and primers with the mutation site. SiRNAs and miRNAs mimics were purchased from GenePharma (Shanghai, China). The sequences were presented in the Supplementary Table 1 . Plasmids, siRNAs and miRNAs transfection were performed by Lipofectamine 3000 (Invitrogen, Waltham, MA, USA), according to the manufacturer’s protocol.
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