Pmirglo dual luciferase reporter plasmid

Manufactured by Promega

Sourced in United States

The PmirGLO Dual-Luciferase Reporter Plasmid is a reporter vector used for gene expression analysis. It contains both Firefly and Renilla luciferase reporter genes, allowing for normalization of experimental results.

Automatically generated - may contain errors

Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Luminoskan ascent, by Thermo Fisher Scientific (3 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Dual glo luciferase assay, by Promega (1 mentions) Kod plus mutagenesis kit, by Toyobo (1 mentions) Lipofectamin rnaimax reagent, by Thermo Fisher Scientific (1 mentions) Renilla luciferase activity, by Promega (1 mentions) Dual luciferase assay, by Promega (1 mentions) Varioskan flash multimode reader, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay, by Promega (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PmirGLO dual-luciferase reporter PmirGLO dual-luciferase plasmid Dual-luciferase reporter plasmid PmirGLO luciferase reporter PmirGLO reporter plasmid PmirGLO luciferase plasmid Luciferase reporter plasmids PmirGLO reporter PmirGLO plasmid Reporter plasmids

9 protocols using pmirglo dual luciferase reporter plasmid

Mapping Mouse PTEN 3'UTR Regulatory Regions

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The 3′-untranslated region (3′UTR) of mouse PTEN was amplified from C57B 6 L mouse genome DNA, and the whole fragment was divided into four segments since it was too long. The four separate fragments were cloned into the pmirGLO dual-luciferase reporter plasmid (Promega, Madison, USA) to construct four plasmids named pmirGLO-PTEN-3′UTR-F1, F2, F3, and F4 using primers shown in
Table 2. The dual-luciferase reporter plasmids and pmirGLO control plasmid were transfected into TSC2
^–/– MEFs using Lipofectamine 3000 transfection reagent, respectively. The transfection mixture was removed and replaced by fresh full DMEM containing 10 nM rapamycin after 4-6 h of transfection. Then, the cells were harvested and lysed 24 h later. The lysates were added to black 96-well plates to measure Renilla luciferase activity using the Dual Luciferase Reporter Gene Assay Kit (11402ES60; Yisheng, Beijing, China). Renilla luciferase activity was regarded as a normalized control, and all data are shown with the luciferase/Renilla ratio.

Table 2 The primers of PTEN-3’UTR construction

Primer	Sequence
F1	5′-tgCTCGAGtgacaccactgactctgatccag-3′
R1	5′-ggGTCGACCGATAGTAGTTGTACTCTTGC-3′
F2	5′-tgCTCGAGgtggtagagttgggattagggc-3′
R2	5′-gaGTCGACGAGGCATTATCCTGTACACGTC-3′
F3	5′-tgCTCGAGaccccgattcagcctcttcaga-3′
R3	5′-tgGTCGACCCCCAAGGACATGAGAATTGTG-3′
F4	5′-gaCTCGAGgtgaagatggcaggatagtgtc-3′
R4	5′-gtGTCGACCTAGTCTTATGTCCATTGGTAGCC-3′

Wan L., Wang Y., Li J., Wang Y, & Zhang H. (2022). Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs: Effect of Akt/mTOR inhibition on PTEN expression. Acta Biochimica et Biophysica Sinica, 54(11), 1637-1647.

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Constructing Murine MR and SGK1 UTRs

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The 3’ UTRs of mouse MR and SGK1 were constructed using Gibson assembly of synthesized gene fragments (Integrated DNA Technologies, Coralville, IA) from the known sequences downloaded from the National Center for Biotechnology Information (NCBI) databases. For SGK1 we used GenBank Accession # NM_001161850 and MR (NR3C2) GenBank Accession # NM_001083906. The MR-UTR construct was engineered with AccI/SbfI and the SGK1-UTR with XhoI/XbaI restriction sequences for incorporation into a digested pMir-Glo dual luciferase reporter plasmid (Promega, Madison, WI). The putative miR-466 binding sites, located at positions 456 and 699 bp away from the termination codon in the MR-UTR and, 868 bp in the SGK1-UTR (see Fig.2) were eliminated and gene fragments assembled (as above) to produce UTRs in which the predicted miR-466 sites were absent. All constructs were sequenced (GeneWiz, South Plainfield, NJ) to verify the correct sequence and desired deletions were incorporated.

Ozbaki-Yagan N., Liu X., Bodnar A.J., Ho J, & Butterworth M.B. (2020). Aldosterone-induced microRNAs act as feedback regulators of mineralocorticoid receptor signaling in kidney epithelia.. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(9), 11714-11728.

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Cloning miR-219-5p and COX-2 3'-UTR

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To insert the sequence of miR-219-5p into the pmiR-Glo dual-Luciferase reporter plasmid (Promega, Madison, WI, USA), a Cloning Kit (Vazyme Biotech, Nanjing, China) was used. The binding-site CUGUUAG was mutated into GACAAUC, which was used as a negative control. A human genomic DNA of Ishikawa cells was extracted to amplify the sequence of 3′-UTR of COX-2. Therefore, pmiR-Glo plasmid containing the 3′-UTR of COX-2 was constructed. The binding-site ACAAUCA was mutated into UGUUAGU, which was also regarded as a negative control. Primers used to generate plasmid were listed in Supplemental Table S4.

Wang Y, & Yin L. (2021). LINC00461 Promoted Endometrial Carcinoma Growth and Migration by Targeting MicroRNA-219-5p/Cyclooxygenase-2 Signaling Axis. Cell Transplantation, 30, 0963689721989616.

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Dual-Luciferase Assay for miRNA Binding

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The 3′UTR of PLEKHO2 (372bp; ENSG00000241839; GeneID:80301) or NR2F2-AS1 fragment (311bp; ENSG00000247809; GeneID:644192) was inserted into the pmiRGLO Dual-luciferase reporter plasmid (Promega, Madison, WI, USA). The mutant plasmids (mt-PLEKHO2 and mt-NR2F2-AS1) were constructed by the KOD-Plus Mutagenesis Kit (Toyobo, Osaka, Japan). All the plasmids were verified by sequencing. PmiRGLO plasmid (wild type or mutant) was co-transfected with miR-106b mimics or NC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48h, the luciferase activities were measured using the Dual-Glo Luciferase Assay (Promega, USA) on Luminoskan Ascent (Thermo, Madison, WI, USA), according to the manufacturer’s instructions. The primers were used as follows: wt-PLEKHO2-F: 5′-GTTTAAACGAGCTCGCTAGCGGGGTATGTTGGAATCCGAAGC-3′; wt-PLEKHO2-R: 5′-TTGCATGCCTGCAGGTCGACAGGCCAGGGCATGTTGCTG-3′; mt-PLEKHO2-F: 5′-TGTAAAGTGCTGTTTACTGAAAGAGAGAAAGGGGGGG-3′; mt-PLEKHO2-R: 5′-TGCAGAAATCTGGGCAGGTCC-3′; wt-NR2F2-AS1-F: 5′-GTTTAAACGAGCTCGCTAGCTGTGAATCAGTAAACGTACTAGA-3′; wt-NR2F2-AS1-R: 5′-TTGCATGCCTGCAGGTCGACTAAAAGTGCTGCCCAAGA-3′; mt-NR2F2-AS1-F: 5′-AAAGTGCTGTAGACCTGCAGGCATGACAGCTGAT-3′; mt-NR2F2-AS1-R: 5′-CCCAAGATTGATTGCTCTGATCTG-3′.

Liu S., An G., Cao Q., Li T., Jia X, & Lei L. (2021). The miR-106b/NR2F2-AS1/PLEKHO2 Axis Regulates Migration and Invasion of Colorectal Cancer through the MAPK Pathway. International Journal of Molecular Sciences, 22(11), 5877.

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Luciferase Assay for TBCK 3'-UTR

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The 3′-UTR region of TBCK or mutated 3′-UTR region of TBCK were cloned into a pmirGLO Dual-Luciferase reporter plasmid (Promega). Caki-1 cells were transfected with the constructed TBCK 3′-UTR-pmirGLO Dual-Luciferase reporter plasmid, miR-1208, or anti-miR-1208 (Genolution, Seoul, Korea) by Lipofectamin RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA). Twenty-four hours post-transfection, luciferase activity assays were performed and results normalized to Renilla luciferase activity (Promega). The experiments were repeated three times.

Kim E.A., Jang J.H., Sung E.G., Song I.H., Kim J.Y, & Lee T.J. (2019). MiR-1208 Increases the Sensitivity to Cisplatin by Targeting TBCK in Renal Cancer Cells. International Journal of Molecular Sciences, 20(14), 3540.

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Confirming SOCS2 as Hsv2-miR-H9-5p Target

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Dual-luciferase reporter assays were conducted to confirm that SOCS2 gene containing the predicted Hsv2-miR-H9-5p binding site was indeed target of Hsv2-miR-H9-5p. The human SOCS2 3′-UTR containing the predicted binding sites for Hsv2-miR-H9-5p was cloned into the pmiR-GLO dual-luciferase reporter plasmid (Promega) downstream of the firefly luciferase coding region as pmiR-GLO-SOCS2-WT (wild type, WT). Corresponding reporters containing mutations in the seed region of Hsv2-miR-H9-5P binding sites were generated through synthesizing DNA sequences containing described mutations (Sangon Biotech), annealing, and then cloning into the pmiR-GLO vector as pmiR-GLO-SOCS2-MUT (mutated type, MUT). HEK293T cells were co-transfected with the appropriate reporter plasmids and Hsv2-miR-H9-5P mimic in a 24-well plate. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent according to the manufacturer's instructions. Cells were lysed 24 h after transfection and analyzed for firefly and Renilla luciferase activities using the Dual-Luciferase Assays (Promega) on a Varioskan Flash Multimode Reader (Thermo Scientific).

Wang X., Liu S., Zhou Z., Yan H, & Xiao J. (2017). A herpes simplex virus type 2-encoded microRNA promotes tumor cell metastasis by targeting suppressor of cytokine signaling 2 in lung cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 39(5).

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GLDC 3'-UTR Luciferase Assay

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A 70-bp fragment of the GLDC 3ʹ-UTR or 3ʹ-UTR mutant sequence was cloned into the pmirGLO dual-luciferase reporter plasmids (Promega, Madison, WI, USA). The Huh7 and PLC cells were cultured on 24-well tissue culture plates at a density of 3 × 10⁴ cells per well, followed by co-transfection with the reporter constructs together with miR-30d-5p-mimic, miR-30d-5p inhibitor, or their corresponding controls using Lipofectamine 3000.

Zhuang H., Wu F., Wei W., Dang Y., Yang B., Ma X., Han F, & Li Y. (2019). Glycine decarboxylase induces autophagy and is downregulated by miRNA-30d-5p in hepatocellular carcinoma. Cell Death & Disease, 10(3), 192.

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Dual-Luciferase Reporter Assay for circAGAP1 and E2F3

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PmirGLO dual-luciferase reporter plasmids (Promega) were used for luciferase reporter analysis. Wild-type circAGAP1 (circAGAP1-WT) and mutant circAGAP1 (circAGAP1-MUT), which had mutations at the potential miR-15a-5p binding sites, were amplified by PCR and cloned into the pmirGlo dual-luciferase vector with restriction sites of SacI and XhoI to construct a luciferase reporter vector (pmirGLO-circ_0015756-WT and pmirGLO-circ_0015756-mut, respectively; GeneChem Co., Shanghai, China). Similarly, WT E2F3 (pmirGLO-E2F3-WT) or E2F3 with mutations at the potential miR-15a-5p binding sites (pmirGLO-E2F3-MUT) were also designed by Shanghai GeneChem Co.
The reporter plasmids were cotransfected with miR-15a-5p mimic or control mimic (mimic NC) with Lipofectamine 3000 (Thermo Fisher Scientific) and cultured further for 48 h. Luciferase activity was analyzed by the dual-luciferase reporter assay (Promega). Experiments were performed in triplicate.

Lv Q., Wang G., Zhang Y., Shen A., Tang J., Sun Y., Ma C, & Wang P. (2021). CircAGAP1 promotes tumor progression by sponging miR-15-5p in clear cell renal cell carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 40, 76.

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Luciferase Assay for miR-15a/16 Targeting GRK2

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293T cells purchased from Stem Cell Bank of Chinese Academy of Sciences (Shanghai, China) were used to perform the luciferase reporter assay. The 293T cells were cultured in DMEM (Gibco, Rockville, MD, USA), containing 10% fetal bovine serum and a 1% penicillin/streptomycin mix, and grown in a humidified atmosphere with 95% air and 5% CO₂ at 37°C. The fragments of GRK2 3′-UTRs harboring the predicted wild-type seed-matched (WT) or mutant (MT) binding sites were inserted into pmirGLO dual-luciferase reporter plasmids (Promega, Madison, WI, USA). The WT or MT constructs were cotransfected with miR-15a/16 mimics (RiboBio, Guangzhou, China) into 293T cells using Lipofectamine 2000 (Invitrogen) as per the protocols of the manufacturer. After incubation for 48 h, the luciferase reporter activity was determined using the dual-luciferases reporter system (Promega).

Li T., Wan Y., Sun L., Tao S., Chen P., Liu C., Wang K., Zhou C, & Zhao G. (2019). Inhibition of MicroRNA-15a/16 Expression Alleviates Neuropathic Pain Development through Upregulation of G Protein-Coupled Receptor Kinase 2. Biomolecules & Therapeutics, 27(4), 414-422.

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