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Dual luciferase reporter plasmid

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The Dual-luciferase reporter plasmid is a laboratory tool used to measure and compare the activity of two different promoters or regulatory sequences. It contains the coding sequences for two distinct luciferase enzymes, which produce bioluminescent light when supplied with their respective substrates. This plasmid allows for simultaneous quantification of the activities of the two reporter genes, enabling researchers to normalize and compare the expression levels of their experimental and control samples.

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Lab products found in correlation

Attractene transfection reagent, by Qiagen (4 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Dual luciferase reporter system, by Promega (2 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (2 mentions) Pgl3 basic, by Promega (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Biotek synergy plate reader, by Agilent Technologies (1 mentions) Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions) Dual glo luciferase assay system, by Promega (1 mentions) 96 well plate, by Corning (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Renilla luminescence, by Promega (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Glomax luminometer, by Promega (1 mentions)

Close spelling variants of this product

Dual-luciferase reporter gene plasmids PsiCHECK2 dual luciferase reporter plasmid PmirGLO dual-luciferase reporter plasmid Luciferase reporter plasmids Reporter plasmids

10 protocols using dual luciferase reporter plasmid

1

Validating miR-1299 Binding Sites

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CircZCCHC2 or TPR 3′UTR sequences containing the wild-type- and mutant miR-1200-binding sites were synthesized and inserted into dual luciferase reporter plasmids (Promega, USA) that included the psiCheck2 promoter. BC cells were co-transfected with the miR-1299 mimics or control mimics. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) after 48 h.
Zhang F., Wei D., Xie S., Ren L., Qiao S., Li L., Ji J, & Fan Z. (2024). CircZCCHC2 decreases pirarubicin sensitivity and promotes triple-negative breast cancer development via the miR-1200/TPR axis. iScience, 27(3), 109057.
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2

Dual Luciferase Assay for miRNA Target Prediction

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To experimentally predict the possible miRNA target genes and binding sequences, the wild-type and mutant sequences were synthesized and cloned into dual luciferase reporter plasmids (Promega) containing the psiCheck2 promoter. After MDA-MB-231 cells were inoculated into a 96-well plate and cultured for 24 h, they were cotransfected with the wild-type or mutant reporter gene plasmids and overexpression or silencing plasmid mimics. Luciferase activity was measured 48 h after transfection.
Wang L., Zhou Y., Jiang L., Lu L., Dai T., Li A., Chen Y, & Zhang L. (2021). CircWAC induces chemotherapeutic resistance in triple-negative breast cancer by targeting miR-142, upregulating WWP1 and activating the PI3K/AKT pathway. Molecular Cancer, 20, 43.
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3

Regulation of PDK4 expression by miR-16-5p

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The promoter (−1 kb) of wild-type PDK4 was amplified by PCR and cloned into the dual-luciferase reporter plasmid (Promega) pGL3-basic to yield pGL3-PDK4 reporter. The reporter was transfected into both parental and Dox-resistant cervical cancer cells seeded in 96-well plates at the density of 1.5 × 104 cells per well using the Attractene Transfection Reagent (catalog number, 301005, QIAGEN). The firefly luciferase activity was measured and normalized to that of Renilla luciferase. To analyze the effect of miR-16-5p on PDK4, we cloned the 3′ UTR of PDK4 into pmirGLO plasmid to generate pmirGLO-PDK4 plasmid. The effect of miR-16-5p on 3′ UTR of PDK4 was assessed by using the Attractene Transfection Reagent (QIAGEN).
Zhao Z., Ji M., Wang Q., He N, & Li Y. (2020). miR-16-5p/PDK4-Mediated Metabolic Reprogramming Is Involved in Chemoresistance of Cervical Cancer. Molecular Therapy Oncolytics, 17, 509-517.
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4

Notch2 3'UTR Luciferase Assay

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The 3′-UTR sequence of wild-type Notch2 and that of a target-site mutant were amplified by PCR, cloned into a dual-luciferase reporter plasmid (Promega, Madison, WI, USA), and named pGL3-Notch2–3′-UTR-WT (wild-type vector) and pGL3-Notch2–3′-UTR- MUT (mutant vector). TE-1 and KYSE410 cells in logarithmic growth phase were inoculated into 96-well plates at 1.5 × 104 cells per well before transfection. TE-1 and KYSE410 cells were co-transfected with the Wt or Mut vector and miR-1 mimics, NC, miR-1 inhibitor, or inhibitor NC using the Attractene Transfection Reagent (Qiagen). The ratio of firefly to Renilla luciferase activity was measured with a dual-luciferase reporter system (Promega, Madison, WI, USA) after transfection for 48 h.
Liu W., Li M., Chen X., Zhu S., Shi H., Zhang D., Cheng C, & Li B. (2018). MicroRNA-1 suppresses proliferation, migration and invasion by targeting Notch2 in esophageal squamous cell carcinoma. Scientific Reports, 8, 5183.
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5

Validation of miR-802 Binding Site in RAN 3'UTR

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The potential targets of miR-802 were analyzed by TargetScan7.0 (http://www.targetscan.org) and miRanda (http://www.microrna.org) softwares. The wild type (WT) RAN and the target site mutant (MT) 3ʹ-UTR sequence were amplified by PCR, cloned into the dual-luciferase reporter plasmid (Promega, Madison, WI, USA) and named as pGL3-RAN-3ʹ-UTR-WT (WT vector) (5ʹ-CTAGAAATTAGGATTAATAAAGATGGCACTTTCCCGTTTTATTCCAGTT-3ʹ) and pGL3-RAN-3ʹ-UTR-MT (MT vector) (5ʹ-CTAGAAATTAGGATTAATAAAGATGTTTGCACCCCGTTTTATTCCAGTT-3ʹ). The mutated putative miR-802 binding site in the RAN 3ʹUTR was generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, Cedar Creek, USA) according to the manufacturer’s protocols. HCT-116 cells in logarithmic growth phase were plated into 96-well plates at 1.5 × 104 cells/well. And then HCT-116 cells were co-transfected with WT or MT vectors and miR-802 mimics or miR-NC using Attractene Transfection Reagent (Qiagen). After 48 hrs of transfection, the ratio of firefly to Renilla luciferase activity was measured using a dual-luciferase reporter system (Promega, Madison, WI, USA).
Feng H., Liu L., Xu L., Wang H., Hua Q, & He P. (2020). MiR-802 Suppresses Colorectal Cancer Cell Viability, Migration and Invasion by Targeting RAN. Cancer Management and Research, 12, 2291-2300.
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6

Dual-Luciferase Assay for miR-29a Targets

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5′-phosphorylated oligonucleotides containing predicted binding sites of miR-29 target genes, 6 bp of flanking DNA, and SacI/XhoI restriction site overhands were annealed then ligated into the dual luciferase reporter plasmid (Promega E1960). For mutated sequences, three to four points were induced in the miR-29a seed binding site using the Q5 site-directed mutagenesis kit (NEB E0554S). Twenty nanograms of recombinant dual-luciferase plasmid and 6 pmol of either miR-29a mimic or scrambled control were mixed with 100 μL of Opti-MEM containing 1 μL of lipofectamine RNAiMax (Invitrogen 13778) in 24-well plates. Reverse transfection was initiated by adding 90,000 HEK-293A cells/well in 500 μL. After 48 h, firefly activity was measured by luminescence and normalized to Renilla activity.
O'Connor S., Murphy E.A., Szwed S.K., Kanke M., Marchildon F., Sethupathy P., Darnell R.B, & Cohen P. (2021). AGO HITS-CLIP reveals distinct miRNA regulation of white and brown adipose tissue identity. Genes & Development, 35(9-10), 771-781.
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7

Quantitative Analysis of Protein Expression

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To assess changes in protein levels, HEK293T cells were transfected
with 12.5 ng dual luciferase reporter plasmid (Promega), 150 ng of the
indicated CIRTS expression vector, and 100 ng of the gRNA expression vector.
About 16 h before transfection, cells were plated on 96-well plates
(Corning) and allowed to grow to 70–80% confluency overnight. The
next day, a total of 20 μL Opti-MEM I Reduced Serum Medium
(ThermoFisher Scientific) per well was used after combining 10 μL
Opti-MEM with 0.5 μL lipofectamine 2000 and 10 μL containing
all the transfection DNA. The solutions were combined and incubated for 15
min before slow addition to cells. After 48 h, luciferase readouts of
firefly luciferase and Renilla luciferase were sequentially measured using
the DualGlo Luciferase Assay System (Promega) on a Biotek Synergy plate
reader according to the manufacturer’s instructions. All experiments
were conducted in at least biological triplicates. Firefly luciferase
luminescence levels were normalized to the corresponding Renilla
luminescence levels to generate the normalized change in protein levels from
the target firefly luciferase gene.
Rauch S., He E., Srienc M., Zhou H., Zhang Z, & Dickinson B.C. (2019). Programmable RNA-guided RNA effector proteins built from human parts. Cell, 178(1), 122-134.e12.
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8

Luciferase Assay for LDHA 3'UTR Regulation

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The 3′‐UTR sequence of wild‐type LDHA and that of a target‐site Mutant (MT) were amplified by PCR, cloned into a dual‐luciferase reporter plasmid (Promega), yielding pGL3‐LDHA–3′‐UTR‐wild‐type (WT) and pGL3‐LDHA‐3′‐UTR‐MT respectively. Cells were inoculated into 96‐well plates at the density of 1.5 × 104 cells per well. Cells were co‐transfected with the WT or MT vector and miR‐33b mimics, NC, miR‐33b inhibitor, or inhibitor NC using the Attractene Transfection Reagent (Qiagen). The ratio of firefly to Renilla luciferase activity was assessed at 48 hours after transfection.
Zhai S., Zhao L., Lin T, & Wang W. (2018). Downregulation of miR‐33b promotes non‐small cell lung cancer cell growth through reprogramming glucose metabolism miR‐33b regulates non‐small cell lung cancer cell growth. Journal of Cellular Biochemistry, 120(4), 6651-6660.
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9

Beclin-1 3'UTR Reporter Assay

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For Beclin-1 3’UTR reporter assay, SY-SH-5Y cells were placed in 24-well plates (1 × 103 cells per well) and then transfected with either psi-CHECKTM2-WT-BECN-3’UTR (wild type) or psi- CHECKTM2-MT-BECN-3’UTR that containing the miR-30c targeting sequence (UGUUUAC) (mutant) dual Luciferase reporter plasmid (Promega, WI, USA) according to manufacture’s protocol. The mimics and inhibitors of hsa-miR-30c and their negative controls (RIBO Bio, Guangzhou, P.R. China) were cotransfected with the reporter plasmids at a final concentration of 100 nmol/μl. Forty–eight hours after transfection in neurons, luciferase activity in lysates was measured with a Dual-Luciferase Reporter Assay System (Promega, WI, USA) followed by the manufacture’s suggestions and normalized against the activity of the pRL-SV40. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI), and the Renilla luciferase activity was normalized to firefly luciferase activity. Independent triplicate experiments were performed for each plasmid construct.
Li L., Jiang H.K., Li Y.P, & Guo Y.P. (2015). Hydrogen sulfide protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord ischemia-reperfusion injury. Journal of Biomedical Science, 22(1), 50.
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10

miR-29 Regulation of LAMC2 in Skin Repair

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Ten thousand HEK293T cells were seeded in Dulbecco's modified Eagle's medium (Life Technologies, Carlsbad, CA) and 10% fetal bovine serum (Gibco) in 24-well plates. The cells were transfected with 20 ng of dual luciferase reporter plasmid (Promega, Southampton, UK) with the 3 0 UTRs for the LAMC2 WT or mutant sequences cloned behind the firefly luciferase gene. Relative luciferase activity was measured after transfection with miR-29 or nonspecific oligonucleotides on a GLOMaX Luminometer (Promega) miR-29eLaminin C2 to Improve Skin Repair
The American Journal of Pathology -ajp.amjpathol.org using Renilla luminescence (Promega) as a control to normalize the level of transfection. The error bars in the images represent the SDs of at least three independent transfections. The Q5 site-directed mutagenesis kit (New England BioLabs, Hitchin, UK) was used to mutate 3 0 UTR of LAMC2 at the site of minimal 6 nucleotides required for miR-29 (seed sequence, UGGUGC) and compared to the levels of the WT 3 0 UTR inhibition by miR-29 or nonspecific oligonucleotides (negative control 1) (Table 1).
Robinson C.J., Thiagarajan L., Maynard R., Aruketty M., Herrera J., Dingle L., Reid A., Wong J., Cao H., Dooley J., Liston A., Müllhaupt D., Hiebert P., Hiebert H, & Kurinna S. (2024). Release of miR-29 Target Laminin C2 Improves Skin Repair. The American journal of pathology, 194(2).
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