Pgl3 reporter plasmid

Manufactured by Promega

Sourced in United States, China

The PGL3 reporter plasmid is a lab equipment product designed for gene expression analysis. It contains a luciferase reporter gene that can be used to measure the activity of a promoter or regulatory sequence of interest. The plasmid is a tool for researchers to study gene expression in various cellular systems.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (4 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Dual luciferase reporter assay kit, by Promega (2 mentions) Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions) Xbai, by New England Biolabs (1 mentions) Lipofectin 2000, by Thermo Fisher Scientific (1 mentions) Ad nc, by Hanbio Biotechnology (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter kit, by Promega (1 mentions) Glomax 20 20 fluorescence detector, by Promega (1 mentions) Protein a agarose, by Merck Group (1 mentions) Pcmv sport6, by Thermo Fisher Scientific (1 mentions) Dual luciferase assay kit, by Promega (1 mentions)

Close spelling variants of this product

PGL3-promoter reporter plasmids PGL3 luciferase reporter plasmid PGL3 basic luciferase reporter plasmid vector PGL3 firefly luciferase reporter plasmid PGL3-basic firefly luciferase reporter plasmid PGL3-basic reporter plasmid PGL3 promoter luciferase reporter plasmid PGL3-basic luciferase reporter plasmid PGL3 luciferase reporter plasmid vector PGL3-control luciferase reporter plasmid

13 protocols using pgl3 reporter plasmid

miR-19a Regulation of IκBα Expression

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For the luciferase assay, the 3′ UTR of IκBα, including the binding site for miR-19a, was amplified from the MGC-803 cells using the following primers: IκBα, forward 5′-AAGGAGGAGGGCAGAATCAT-3′ and reverse, 5′-ATCTGCATGGTGATGTTGGA-3′. The PCR product was then digested with XbaI (New England Biolabs, Beverly, MA, USA) and cloned into the pGL3 reporter plasmid (Promega Corporation, Madison, WI, USA), downstream of the luciferase reporter gene.
The modified firefly luciferase vector (500 ng/μl) was transfected into HEK293 cells (2×10⁵ cells/ml), as described previously, and firefly and Renilla luciferase activities were measured 48 h after transfection using a Dual-Luciferase Reporter Assay system (Promega Corporation). Firefly activity was normalized to Renilla activity to control the transfection efficiency.

YANG F., WANG H., JIANG Z., HU A., CHU L., SUN Y, & HAN J. (2015). MicroRNA-19a mediates gastric carcinoma cell proliferation through the activation of nuclear factor-κB. Molecular Medicine Reports, 12(4), 5780-5786.

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Validating miR-124 target in 786-O

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The 3'-UTR of CAPN4 (miR-124 binding site) was synthesized and subcloned into a pGL3 reporter plasmid (Promega, USA). The plasmid was co-transfected with miR-124 mimics or miR-NC into 786-O cells for 48 h. 786-O cells were collected and the activity of luciferase was measured using the dual-luciferase reporter assay kit (Promega, USA).

Mao Q., Zhuang Q., Shen J., Chen Z., Xue D., Ding T, & He X. (2021). MiRNA-124 regulates the sensitivity of renal cancer cells to cisplatin-induced necroptosis by targeting the CAPN4-CNOT3 axis. Translational Andrology and Urology, 10(9), 3669-3683.

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Luciferase Reporter Assay for FOXA2

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The Dual Luciferase Reporter Assay System (Promega, USA) was used for luciferase reporter assay, for searching the target gene for miR-92a. The entire 3’-UTR of human FOXA2 was amplified using PCR. The PCR product was inserted into the p-GL3 -reporter plasmid (Promega) and confirmed by sequencing. The mutant of FOXA2 3’-UTR (from CCCACC to CCCAGC) was used to test the binding specificity, and FOXA2 3’-UTR mutant form was also inserted into the equivalent luciferase reporter. HepG2 cells were cultured in 24-well plates. The plasmids and 100 pmol of miR-92a or miR-control were co-transfected with the Renilla plasmid using Lipofectamine 2000 (Invitrogen) into HepG2 cells, and 48 hr post-transfection, the cells were assayed. The promoter activity was calculated as firefly/Renilla.

Wang L., Wu J, & Xie C. (2017). miR-92a promotes hepatocellular carcinoma cells proliferation and invasion by FOXA2 targeting. Iranian Journal of Basic Medical Sciences, 20(7), 783-790.

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Luciferase Assay for miR-17-5p Targeting

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The MAP3K8 3′UTR segment containing the putative miR-17-5p target sites was subcloned into the pGL3 reporter plasmid (Promega). A mutant construct was also generated by PCR-based mutagenesis using mutagenic primers. The luciferase reporter and hsa-miR-17-5p expressing or mock plasmids were co-transfected with the Renilla luciferase transfection control plasmid. Luciferase reporter activity was measured 48 hours after transfection with the Dual-Luciferase Reporter Assay System (Promega).

Tsubota A., Mogushi K., Aizaki H., Miyaguchi K., Nagatsuma K., Matsudaira H., Kushida T., Furihata T., Tanaka H, & Matsuura T. (2014). Involvement of MAP3K8 and miR-17-5p in Poor Virologic Response to Interferon-Based Combination Therapy for Chronic Hepatitis C. PLoS ONE, 9(5), e97078.

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Overexpression of AK006774 Using Adenoviruses

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Adenoviruses overexpressing AK006774 (Ad-AK006774) and control vector (Ad-nc) were synthesized by Hanbio (Shanghai, China). The miR-448 mimic and inhibitor, along with their negative controls, were synthesized by General Bio Company (Hefei, Anhui). Wild-type and mutant sequences containing the binding sites of miR-448 targeting AK006774 and bcl-2 were synthesized and cloned into the pGL3 reporter plasmid (Promega, USA). These oligonucleotides were transfected into cells using Lipofectin2000 (Thermo Fisher Scientific), USA kit according to the instructions [14 (link)].

Nie S., Cui X., Guo J., Ma X., Zhi H., Li S, & Li Y. (2021). Long non-coding RNA AK006774 inhibits cardiac ischemia-reperfusion injury via sponging miR-448. Bioengineered, 12(1), 4972-4982.

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PDK Transactivation by Sima

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To measure the transactivation activity of Sima on the PDK gene, the promoter region was subcloned into the pGL3 reporter plasmid (Promega, USA) using the following primers: PDK promoter F (CGC ACG CGT CCA ACA CTT GTA GTG GTT AAA AGT GTA G) and R (CGC AGA TCT CTC ACT CTC TTC GCA GGA CGT TG). PDK reporters with HRE mutations were generated using the QuikChange^TM site-directed mutagenesis kit (Agilent Technologies, USA) with the following primer pairs: HRE1 mutant F (ACA CTG CGG CTC TCC CGA TCA AAA GCG CTC GCA GC) and R (GCT GCG AGC GCT TTT GAT CGG GAG AGC CGC AGT GT); and HRE2 mutant F (CCC GCC TTT CAA CAC AAA AAC AAC AAC AAC AAA TCG GAG CAG AGC TAA CAC AA) and R (TTG TGT TAG CTC TGC TCC GAT TTG TTG TTG TGT TGA AAG GCG GG). S2 cells were transfected with the wild-type or HRE mutant PDK reporters, pUAST-Sima, pR-TK Renilla reporter, and pMT-GAL4 plasmids. Two days post-transfection, Sima expression was induced by CuSO4 treatment. After 24 h, the luciferase assay was performed using the Dual-Luciferase^TM Reporter Assay Kit (Promega) according to the manufacturer's instructions. The results are expressed as average luciferase activity ± SD from three independent experiments.

Lee Y., Kim J., Kim H., Han J.E., Kim S., Kang K.H., Kim D., Kim J.M, & Koh H. (2022). Pyruvate Dehydrogenase Kinase Protects Dopaminergic Neurons from Oxidative Stress in Drosophila DJ-1 Null Mutants. Molecules and Cells, 45(7), 454-464.

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Validating miR-1306-5p Regulation of HSD17B7

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The potential binding sites between miR-1306-5p and HSD17B7 were predicted with Starbase (http://starbase.sysu.edu.cn/index.php). Luciferase reporter gene assay confirmed this assumption. The wild type or muted 3’UTRs of HSD17B7 was inserted into the pGL3-reporter plasmid (Promega, Madison, WI, USA), and the luciferase reporter plasmids HSD17B7 3’UTRs-WT and HSD17B7 3’UTRs were constructed. Next, the HSD17B7 3’UTRs or HSD17B7 3’UTRs-WT and miR-1306-5p mimic or a corresponding negative control (NC mimic) were co-transfected into HEK-293T cells with Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) when grown to approximately 70% confluence. After transfection for 24 hours, we harvested the cells and analyzed them with a dual luciferase reporter kit (Promega, Madison, Wisconsin, USA) to standardize luciferase reporter activity to Renilla luciferase activity.

Wang D., Li Y., Zhai Q.Q., Zhu Y.F., Liu B.Y, & Xu Y. (2021). Quercetin ameliorates testosterone secretion disorder by inhibiting endoplasmic reticulum stress through the miR-1306-5p/HSD17B7 axis in diabetic rats. Bosnian Journal of Basic Medical Sciences, 22(2), 191-204.

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Mouse Pparg2 Promoter Luciferase Assay

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Luciferase reporter genes driven by mouse Pparg2 promoters were constructed by insertion of PCR‐amplified promoters into pGL3 reporter plasmid (Promega, Madison, WI). All plasmids and transfection assays used in this study have been described.15 HeLa and Hepa1‐6 were maintained in Dulbecco's modified Eagle's medium plus 10% bovine growth serum. A chromatin immunoprecipitation (ChIP) assay was also performed to identify recruitment of HNF4α and HES6 to the Pparg2 promoter region using mouse liver as described.15

Park J.E., Lee M., Kim S., Zhang Y., Hardwick J.P, & Lee Y.K. (2017). Hairy and enhancer of split 6 prevents hepatic lipid accumulation through inhibition of Pparg2 expression. Hepatology Communications, 1(10), 1085-1098.

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CncC Transactivation of IDH Promoter

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To measure transactivation activity of CncC on the IDH gene, the promoter and 5’ untranslated region (S1C Fig) were subcloned into pGL3 reporter plasmid (Promega) using following primers: IDH promoter F (gcg ggt acc cag tta ttc gct gcg tct gat tgg) and IDH promoter R (gcg gga tcc gaa ccg acc gac gac tgg aaa cg). For generating the IDH reporter with ARE mutation, the first five bases (TGACG) of the putative ARE (TGACGGGGC) were deleted by QuikChange^™ site directed mutagenesis kit (Agilent Technologies). S2 cells were transfected with wild type or ARE mutant IDH reporter, pUAST-CncC, pRL-TK Renilla reporter, and pMT-GAL4 plasmids. Two days later, CncC expression was induced by CuSO4 treatment. After 24 h, luciferase assays were performed using Dual-Luciferase^™ reporter assay kit (Promega) according to the manufacturer's instructions. The average luciferase activity with standard deviation was obtained from three independent experiments.

Yang J., Kim M.J., Yoon W., Kim E.Y., Kim H., Lee Y., Min B., Kang K.S., Son J.H., Park H.T., Chung J, & Koh H. (2017). Isocitrate protects DJ-1 null dopaminergic cells from oxidative stress through NADP+-dependent isocitrate dehydrogenase (IDH). PLoS Genetics, 13(8), e1006975.

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Characterizing miR-31-5p Regulatory Interactions

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We PCR amplified the miR-31-5p sequence containing binding sites for wild-type MAGI2-AS3 and TNS1 according to the Starbase and cloned into the pGL3 reporter plasmid (Promega, Beijing, China). Then we constructed target plasmids containing wild-type or mutant MAGI2-AS3 and TNS1 constructs (Thermo Fisher Scientific, Shanghai, China). We cultured T24 cells in a 6-well plate for 24 h, and then transfected them with different combinations of reporter and target plasmids for 48 h. We used the GloMax 20/20 fluorescence detector (Promega, Beijing, China) to detect the fluorescence intensity of the reporter gene plasmid.

Tang C., Cai Y., Jiang H., Lv Z., Yang C., Xu H., Li Z, & Li Y. (2020). LncRNA MAGI2-AS3 inhibits bladder cancer progression by targeting the miR-31-5p/TNS1 axis. Aging (Albany NY), 12(24), 25547-25563.

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