Pmirglo control vector

Manufactured by Promega

Sourced in United States, Italy

The PmirGLO control vector is a plasmid designed for use as a control in luciferase reporter gene assays. The vector contains a constitutive promoter that drives the expression of the firefly luciferase gene. It can be used as a positive control to verify the functionality of luciferase-based reporter gene assays.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions) Dual luciferase reporter assay kit, by Promega (3 mentions) Dual luciferase assay system, by Promega (2 mentions) Dual luciferase assay, by Promega (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions) Polyethyleneimine, by Polysciences (1 mentions) Luciferase assay kit, by Promega (1 mentions) Quickchange xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Fugene transfection reagent, by Promega (1 mentions) Quikchange site directed mutagenesis kit, by Promega (1 mentions) Dual luciferase assay kit, by Yeasen (1 mentions) Kapa probe fast qpcr master mix, by Roche (1 mentions) Lightcycler 480 system, by Roche (1 mentions) Luciferase activity assay kit, by Beyotime (1 mentions) Mirna mimics or inhibitor, by GenePharma (1 mentions) Microplate reader, by Molecular Devices (1 mentions) Dual luciferase reporter system, by Promega (1 mentions) Mir 346 mimic, by GenePharma (1 mentions) Mir 346 inhibitor, by GenePharma (1 mentions)

18 protocols using pmirglo control vector

Regulation of Atg16l1 by miR-223

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Mir223 mimics, control mimics (Mim-CN), Mir223 inhibitor (a Mir223-specific antagomir), and control inhibitor (In-CN) were synthesized by Ribobio (Guangzhou, China). For the dual-luciferase assay, the MRE found in the 3’ UTR region of the Atg16l1 mRNA and its mutated version were cloned into a pmirGLO-control vector (Promega, E1741) at the 3’ region of the luciferase gene using the following linker primers: Atg16l1 wild-type primers. 5’-CGTAGCGGCCGCTAGTTGGGGAGTACAGGGTCCCT-3’ and 5’-CTAGAGGGACCCTGTACTCCCCAACTAGCGGCCGCTACGAGCT-3’ and Atg16l1 mutant primers 5’-CTAGAGGGACCCTGTACTCCCCAACTAGCGGCCGCTACGAGCT-3’ and 5’-CTAGAGGGTGGGAGTACTCCCCAACTAGCGGCCGCTACGAGCT-3’.
Transient transfection of BV2 cells was performed using either polyethyleneimine (Polysciences Inc., 23,966) or Lipofectamine® RNAi Max reagent (Invitrogen, 13,778–075) according to the manufacturer’s instructions. Briefly, 293T cells were harvested and lysed 48 h post-transfection. Firefly and Renilla luciferase activities were measured using a dual-luciferase-reporter assay system (Promega, E1910). The results were evaluated through normalization of the firefly luciferase activity with the Renilla luciferase activity.

Li Y., Zhou D., Ren Y., Zhang Z., Guo X., Ma M., Xue Z., Lv J., Liu H., Xi Q., Jia L., Zhang L., Liu Y., Zhang Q., Yan J., Da Y., Gao F., Yue J., Yao Z, & Zhang R. (2018). Mir223 restrains autophagy and promotes CNS inflammation by targeting ATG16L1. Autophagy, 15(3), 478-492.

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Validating MUC13 as miR-145 Target

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Dual-luciferase 3′ UTR reporter assay was carried out to validate MUC13 as a direct target of miR-145. The luciferase-UTR reporter constructs were generated by inserting the MUC13 3′ UTR carrying a putative miR-145 binding site into pmirGLO control vector (catalog number E1330; Promega). Additionally, in the wild type MUC13 3′ UTR (WT-MUC13 3′ UTR), a mutant MUC13 3′ UTR (MUT-MUC13 3′ UTR) reporter construct was made by site-directed mutagenesis in the putative target site of miR-145 using Quickchange XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). All PCR products were verified by DNA sequencing. HPAF-II cells were transiently co-transfected with reporter plasmids (1μg) and 100 nM of miR-145 or miR-NC for 48 h. Luciferase assays were performed using a luciferase assay kit (catalog number E2940; Promega) according to the manufacturer's protocol. The normalized luciferase activity was expressed as a ratio of firefly luciferase to Renilla luciferase units.

Khan S., Ebeling M.C., Zaman M.S., Sikander M., Yallapu M.M., Chauhan N., Yacoubian A.M., Behrman S.W., Zafar N., Kumar D., Thompson P.A., Jaggi M, & Chauhan S.C. (2014). MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer. Oncotarget, 5(17), 7599-7609.

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Luciferase Assay for Gli1 Regulation

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CP-A and CP-B cells were plated onto 96-well plates and cultured overnight before cotransfection with 2 ng pRL-TK and 20 ng Gli1-pGL3, a luciferase reporter driven by Gli1 promoter (Gli1 promoter regions, -979 to 33 nt) or the pGL3-Basic vector with FuGene transfection reagent (Promega). After transfection, cells were treated with omeprazole or DMSO. After 48 h, cells were harvested and the luciferase activity was determined using the Dual-Luciferase Reporter Assay Kit (Promega).
To construct an expression vector containing the Gli1 3′-UTR fused to the 3′-end of a luciferase reporter, a 219-bp fragment containing the predicted miR-203a-3p target sites was synthesized and ligated into the pmir-Glo-control vector (Promega, United States). The 3′-UTR of Gli1 containing one putative miR-203a-3p-binding site was amplified and cloned into a pmir-Glo control vector with the restriction endonucleases NheI/SalI. In the mutated fragment, eight bases were introduced into the predicted miR-203a-3p target sites. Cells were plated onto 96-well plates 24 h before treatments. After 48 h, cells were harvested and the luciferase activity was determined as described above. All results were expressed as the relative firefly luciferase activity normalized to Renilla luciferase activity.

Hou Y., Hu Q., Huang J, & Xiong H. (2018). Omeprazole Inhibits Cell Proliferation and Induces G0/G1 Cell Cycle Arrest through Up-regulating miR-203a-3p Expression in Barrett’s Esophagus Cells. Frontiers in Pharmacology, 8, 968.

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SphK2 3'UTR Luciferase Assay Protocol

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The 3′-UTR of the SphK2 fragment was PCR-amplified from human genomic DNA and inserted into the pmiR-GLO control vector (Promega, Madison, WI) at the XhoI and XbaI sites 3′ to the luciferase gene. Primer sequences used for PCR amplification were as follows: forward 5′-AUGGGACCAGACGUGAUGCUGGA-3′, reverse 5′-GUUGUUUUAGUGACUACGACCU-3′. The 3′-UTR of SphK2 was confirmed with sequencing and named pmiR-GLO-WT. Site-directed mutagenesis of the miR-338-3p target site in the SphK2 3′-UTR (pmiR-GLO-mut) was carried out using a Quikchange site-directed mutagenesis kit (Promega, Madison, WI), with pmiR-GLO-WT as the template. For the luciferase reporter assay, A549 and H1299 cells were cultured in 96-well plates. Then, using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), they were each cotransfected with wildtype or mutant reporter plasmid (100 nM) and microRNA (100 nM). At 48 h after transfection, luciferase activity was measured using the dual-luciferase assay system (Promega, Madison, WI).

Zhang G., Zheng H., Zhang G., Cheng R., Lu C., Guo Y, & Zhao G. (2017). MicroRNA-338-3p suppresses cell proliferation and induces apoptosis of non-small-cell lung cancer by targeting sphingosine kinase 2. Cancer Cell International, 17, 46.

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Ccn2 3'-UTR Interaction Validation

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The predicted 3′-UTR sequence of Ccn2 interacting with miR-15b-5p/miR-290a-5p and mutated sequences within the predicted target sites were synthesized and inserted into the pmirGLO control vector (Promega). The insert sequences used for the luciferase reporter assay are listed in Supplementary Table 10. HEK293T cells were transfected with 50 nM miR-15b-5p/miR-290a-5p or negative control and 400 ng of the wild-type or mutant 3′UTR plasmid by Lipofectamine 2000 (Invitrogen). After 48 h of transfection, the luciferase activity of the cells was measured using a Dual Luciferase Assay Kit (Yeasen). Renilla luciferase was used to normalize the value of firefly luciferase.

Yu L., Wen H., Liu C., Wang C., Yu H., Zhang K., Han Q., Liu Y., Han Z., Li Z, & Liu N. (2023). Embryonic stem cell-derived extracellular vesicles rejuvenate senescent cells and antagonize aging in mice. Bioactive Materials, 29, 85-97.

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Investigating p21 Regulation by miR-208b

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The p21 was amplified from human genomic DNA by PCR, containing putative binding sites for miR-208b, attached to the pmirGLO control vector (Promega, Madison, WI, USA). The 3′UTR of p21 was named pmirGLO-Wt, and pmirGLO-Wt served as the template to occur the pmirGLO-Mut plasmid. Luciferase activity was measured by the dual luciferase assay system (Promega, Madison, WI, USA).

Li C., Chen X., Huang J., Sun Q, & Wang L. (2015). Clinical impact of circulating miR-26a, miR-191, and miR-208b in plasma of patients with acute myocardial infarction. European Journal of Medical Research, 20(1), 58.

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Overexpression of miR-130b-5p and CCNG2 Regulation

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miR-130b-5p was cloned into a lentiviral vector PreMiR-130b (System Biosciences, Mountain View, CA, USA) that was used to overexpress the miRNA in MDA-MB-231 cells. Expression of miR-130b-5p was verified and quantified using KAPA PROBE Fast qPCR Master Mix (Kapa Biosystems, Boston, MA, USA), and the LightCycler 480 System (Roche, Basel, Switzerland).
A CCNG2 luciferase reporter construct was made by introducing the CCNG2 3′-UTR carrying a predicted miR-130b-5p binding site (5′-CCTTGGAGATACTGAAAGAGA-3′) into the pmirGLO control vector (Promega, Madison, WI, USA). Site-directed mutagenesis of the putative miR-130b-5p binding site was made using a facile PCR procedure [40 (link)]. All PCR products were verified by DNA sequencing before use.

Chang Y.Y., Kuo W.H., Hung J.H., Lee C.Y., Lee Y.H., Chang Y.C., Lin W.C., Shen C.Y., Huang C.S., Hsieh F.J., Lai L.C., Tsai M.H., Chang K.J, & Chuang E.Y. (2015). Deregulated microRNAs in triple-negative breast cancer revealed by deep sequencing. Molecular Cancer, 14, 36.

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3'-UTR LIP mRNA Luciferase Assay

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The 3′-UTR LIP mRNA wild-type and mutated sequences (GenBank accession No. MT176433) were cloned into the pmirGLO control vector (Promega, Madison, WI, USA) to obtain the pmirGLO-lip-3′-UTR-WT/MUT plasmids, respectively. The same sequences were cloned into the pEGFP-C1 vector to obtain the pEGFP-lip-3′-UTR-WT/MUT plasmids, respectively.
HEK293 cells were seeded on 48-well plates overnight before transfection and then transfected with 25 ng of dual-luciferase reporter WT/MUT vector and 50 nM of miRNA mimics or inhibitor (GenePharma, Suzhou, CHN). Dual-luciferase reporter assays were performed 24 h after transfection using the luciferase activity assay kit, following the manufacturer’s instructions (Beyotime, Beijing, CHN). Firefly luciferase activity was normalized to Renilla luciferase activity. Relative light unit (RLU) data were measured using a microplate reader (Molecular Devices, San Jose, CA, USA) in luminescence detection mode.

Ma L., Gou M., Du Z., Zhu T., Li J., Li Q.W, & Pang Y. (2021). MicroRNA expression profile in Lampetra morii upon Vibrio anguillarum infection and miR-4561 characterization targeting lip. Communications Biology, 4, 995.

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miR-205 Regulation of RUNX2 Expression

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Using the online prediction algorithm (TargetScan version 7.2; http://www.targetscan.org/vert_72/), it was identified that the 3′-UTR of RUNX2 contains a putative binding sequence of miR-205. The wild-type (wt) RUNX2 3′-UTR or mutant (mut) RUNX2 3′-UTR sequences were cloned into a pmirGLO control vector (Promega Corporation, Madison, WI, USA). Cells were co-transfected with either wt RUNX2 3′-UTR or mut RUNX2 3′-UTR and miR-205 mimic or miR-con using Lipofectamine 2000 according to the manufacturer's protocol. At 48 h following transfection, cells were harvested and luciferase activity relative to the Renilla luciferase activity was measured using a Dual Luciferase Reporter system (Promega Corporation) according to the manufacturer's protocol.

Zhuang L., Guo J., Yao Y, & Li Z. (2018). miR-205 targets runt-related transcription factor 2 to inhibit human pancreatic cancer progression. Oncology Letters, 17(1), 843-848.

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Validation of miR-346 Regulation of SMYD3

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DNA sequences containing the miR-346 binding site on the 3’-UTR of SMYD3 were cloned into the downstream of the firefly luciferase stop codon in a pmirGLO control vector (Promega, Milan, Italy). SMMC-7721 cells were implanted in 24 well plates. The following day, the cells were respectively co-transfected with 25 nM of miR-346 mimic, miR-346 inhibitor or miR-NC (GenePharma, Shanghai, China) and 500 ng of 3’-UTR (WT or Mut) of SMYD3 pmirGLO recombinant vectors. Then the cells were harvested 24h after transfection. Firefly luciferase activity was detected with Dual Luciferase Assay (Promega, Milan, Italy) in accordance with the manufacturer’s instructions.

Zhu W., Qian J., Ma L., Ma P., Yang F, & Shu Y. (2017). MiR-346 suppresses cell proliferation through SMYD3 dependent approach in hepatocellular carcinoma. Oncotarget, 8(39), 65218-65229.

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