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Luciferase reporter vector

Manufactured by Promega
Sourced in United States

The Luciferase reporter vector is a molecular biology tool used to measure gene expression. It contains a luciferase gene that can be fused to a promoter of interest, allowing the quantification of promoter activity through luminescence detection.

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49 protocols using luciferase reporter vector

1

Functional Characterization of IL-6R 3'UTR

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Human genomic DNA was used for amplification of full-length 3′-UTR of IL-6D by PCR and the clone into luciferase reporter vector (Promega, Madison, WI). The Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce the mutations into the putative binding sites at 91–99 bp and 621–627 bp of the 3′UTR of IL-6R. The reporter constructs and miR-negative control or miR-125a mimic were used for co-transfection of renal mesangial cells. The cells were incubated for 24 h, and then the activities of luciferase were determined in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instructions.
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2

CATIP-AS1 and Smad4 Luciferase Assay

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Mutated and wild-type CATIP-AS1 and Smad4 sequences were cloned into the luciferase reporter vector (Promega). BCPAP and BHT-101 cells were co-transfected with the luciferase reporter vectors comprising CATIP-AS1 and Smad4 sequences, and miR-515-5p and miR-negative controls. After 48 hours, the relative luciferase activity was measured with a luciferase assay kit (Promega) in accordance with the manufacturer’s guide. Renilla luciferase activity was invoked as the normal control [30 (link)].
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3

miR-22-3p Regulation of AC016405.3 and ERBB3

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The AC016405.3 and ERBB3 fragments containing binding site for miR‐22‐3p were cloned into the luciferase reporter vector (Promega) (AC016405.3 WT and ERBB3 WT). Mutation (Mut) sites were also designed (AC016405.3 Mut and ERBB3 Mut). By using Lipofectamine 2000, cells were co‐transfected with the luciferase reporter vector and miR‐22‐3p mimics. Mimics NC was served as negative control. 48h later, the cells were collected to detect the activities of firefly and Renilla luciferase. Relative luciferase activity was represented as the ratio of Renilla/firefly.
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4

miR-14 Target Gene Validation

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TargetScan program was employed for prediction of target gene of miR-14. The 3′-UTR of candidate gene was cloned into luciferase reporter vector according to the provider’s instruction (Promega). Both M14 and MV3 cells were seeded into six-well plate the day before transfection. Co-transfection of luciferase reporter plasmid and miR-14 was performed with Lipofectamine 2000 (Invitrogen). Relative luciferase activity was measured by micro-plate reader (Molecular Devices, Sunnyvale, CA).
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5

Validation of miR-4735-3p Binding to SCL40A1

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The potential binding site of miR-4735-3p in SCL40A1 3′-UTR was obtained from TargetScan (http://www.targetscan.org/vert_71/), and then, the predicted sequence was cloned into the luciferase reporter vector (Promega, Madison, WI, USA). HEK293T cells were cotransfected with the miR-4735-3p mimic and luciferase reporter plasmid, and relative luciferase reporter activity was determined at 48 h using Dual Luciferase Reporter Assay System (Promega) [41 (link), 42 (link)].
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6

Validating miR-34a Targeting of GAS1

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Using target scan software, GAS1was predicted to be a target of miR-34a. The full length 3’-UTR of GAS1 was amplified by PCR from mice genomic DNA and cloned at the SacI and XhoI sites into luciferase reporter vector (Promega, Madison, WI, USA). The mutant or WT construct of GAS1 3’UTR was generated by GuangZhou Ribobio Company in China. 293T cells were co-transfected with a reporter construct (pmiR-null Report plasmid, pmiR-GAS1 3’-UTR, pmiR-GAS1 3’-UTR-Mut) and miR-34amimic or miR-negative control. After 24 h of incubation, the luciferase activities were measured in a luminometer with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s recommendations.
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7

Investigating miR-222 Gene Regulation

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The synthesized promoter regions (−1000 bp and −500 bp) of the miR-222 gene were subcloned into the luciferase reporter vector (Promega, USA). Luciferase reporter constructs were packed with an adenoviral system and then cotransfected into NRCMs with a control plasmid, followed by the indicated stimulation: Ad-HMGA1 transfection or siHMGA1 for 48 h. Then, the cells were harvested and lysed, and the luciferase activity was determined with the Dual-Luciferase Reporter Assay Kit (Promega, USA) according to the manufacturer’s instructions. The PGL3 basic vector was used as a negative control.
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8

Ebf1 3'-UTR Luciferase Assay

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Full length of Ebf1 3′-UTR was amplified by PCR and cloned into luciferase reporter vector (Promega, USA) and site mutations were generated using QuickChange Site-directed mutagenesis kit (Stratagen, USA). MLE-15 cells were transfected with luciferase reporter vectors, together with miR-598 mimics or negative control. At 48 h after transfection, cells were lysed and the relative luciferase activity was measured using the Dual-Luciferase Reporter Assay kit (Promega, USA).
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9

Elucidating Transcriptional Regulation by RUNX3 and Suv39H1

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A549 and H1299 cells were co-transfected with RUNX3-OE plasmid or empty vector control and Suv39H1-WT or -MUT. Lipofectamine 2000 (Invitrogen) was used for transfection. After 48 h of transfection at 37 °C with the luciferase reporter vector (Promega), a Dual Luciferase Reporter Assay kit (Promega) was used to evaluate the relative luciferase activities. The Renilla luciferase reporter was used as internal control. The activities of firefly luciferase and Renilla luciferase were quantified by using the dual luciferase reporter assay system (Promega).
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10

Validating miR-96-5p targeting of DTL

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Using TargetScan 7.2 software, DTL was predicted to be a target gene of miR-96-5p. The full-length 3′ untranslated region (UTR) of DTL was amplified by PCR and cloned at the SacI and XhoI sites into a luciferase reporter vector (Promega, Madison, WI, USA). The mutant or wild-type construct of DTL 3′UTR was constructed by RiboBio (RiboBio Co., Guangzhou, China). HEK293T cells in 96-well plates were co-transfected with a reporter construct (pmiR-null Report plasmid, pmiR-DTL 3′UTR-wt, pmiR-DTL 3′UTR-mut) and miR-96-5p mimics or miR-negative control (NC) using Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA). A dual-luciferase reporter assay system (Promega, Madison, WI, USA) was used to measure the levels of luciferase activity after 48 h. The results were normalized by dividing the firefly luciferase activity by the Renilla luciferase activity in accordance with the manufacturer’s instructions.
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