Wild-type LINC01140 (LINC01140-WT) or RGS2 3'UTR (RGS2-WT) sequences with miR-452-5p binding sites were inserted into pGL3 reporter constructs (Promega). Mutant LINC01140 (LINC01140-MUT) or RGS2 (RGS2-MUT) were obtained with the QuikChange site-directed mutagenesis kit (Stratagene, USA). Lipofectamine 2000 was then utilized to deliver the reporter constructs, along with a mimic-NC or miR-452-5p mimic, into the HCC1937 and MDA-MB-231 cells. Forty-eight hours later, the luciferase activities of the different vectors were revealed by the dual-luciferase report analysis system (Promega, USA).
Dual luciferase report analysis system
Manufactured by Promega
Sourced in United States
The Dual-Luciferase Reporter Assay System is a tool for analyzing gene expression in cells. It uses two different luciferase reporter enzymes to simultaneously measure the activities of a primary reporter and an internal control reporter within a single sample. This system provides a rapid, quantitative analysis of gene expression.
Automatically generated - may contain errors
9 protocols using dual luciferase report analysis system
1
Luciferase Reporter Assay for miR-452-5p Targets
2
Luciferase Reporter Assay for miR-148b-3p Targeting PDK4
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PDK4 fragments containing miR-148b-3p binding sites were cloned into pmirGLO oligosaccharide enzyme vectors (Promega, Madison, WI, USA), and then pmirGLO-PDK4-wild type (Wt) reporting vectors were constructed. Based on pmirGLO-PDK4-Wt sequence, a pmirGLO-PDK4 mutant (mut) report vector was constructed using a mutant binding site of miR-148b-3p. The constructed vector was transfected into 293T cells, followed by miR-148-3p and miR-NC, respectively. After 48 h, luciferase activity was measured using a dual-luciferase report analysis system (Promega), and relative activity was expressed as the ratio of luciferase activity in fireflies to Renilla luciferase activity.
3
Luciferase Assay for CASC9-miR-423a Interaction
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We individually inserted wild-type CASC9 and mutant CASC9 into pmirGLO reporter vectors (Promega, Madison, WI, USA). We co-transfected human dermal microendothelial cells (HDECs) with miR-423a-5p mimics and wild-type (WT) CASC9 or mutant (MUT) CASC9 using Lipofectamine 2000 (Invitrogen). We measured the relative luciferase activity 48 h after transfection on the dual- luciferase report analysis system (Promega). The data were expressed as the ratio of Renilla luciferase activity to firefly luciferase activity. Luciferase report analysis was performed to verify the direct binding of miR-423a-5p to SOX12 3ʹ-UTR as described above.
4
Luciferase Assay for DANCR and AKT2 Regulation
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The target site sequence (WT) and the sequence (MUT) after targeted mutation of the WT target site of DANCR and AKT2 mRNA 3′-UTR region were synthesized. Restriction enzymes were adopted to digest pmiR-RB-REPORTTM plasmid (RiboBio, Guangzhou, China), and the artificially synthesized target gene fragments WT and MUT were inserted into pmiR-RB-REPORTTM vector, respectively. The luciferase reporter plasmids WT and MUT that were sequenced correctly were used for subsequent transfection. The vectors containing MUT and WT were co-transfected into HEK293T cells with mimic-NC or miR-194 mimic, respectively. After 48 h of transfection, the cells were collected and lysed. The Renilla luciferase detection kit (YDJ2714, Shanghai Yuduo Biological Technology) was used to determine relative luciferase unit. With firefly luciferase as an internal reference, luciferase activities were analyzed using a dual luciferase report analysis system (Promega Co, Madison, WI).
5
Dual-Luciferase Assay for miR-1294 Binding
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Wild-type hsa_circ_0017842 (WT-circ) or SPARC (SPARC-WT) containing binding sites for miR-1294 were constructed by Beijing Qualityard Biotechnology Co., Ltd. (Beijing, China) using a pGL3 vector (Promega, Madison, USA). Mutant hsa_circ_0017842 (MUT-circ) or SPARC (SPARC-MUT) without binding sites for miR-1294 were also constructed by Beijing Qualityard Biotechnology Co., Ltd. using a pGL3 vector. The aforementioned vectors were co-transfected into GC cells with miR-1294 mimic or mimic-NC. Luciferase activity was detected 48 h after transfection with the aid of a dual-luciferase report analysis system (Promega).
6
Validation of BCYRN1-miR-490-3p-POU3F2 Axis
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Associations between BCYRN1 and miR-490-3p, as well as miR-490-3p and POU3F2 were affirmed by dual luciferase assay. Firstly, Wild Type (WT)-BCYRN1, Mutant (MUT)-BCYRN1, WT-POU3F2, or MUT-POU3F2 was cloned into luciferase vector pGL3. Subsequently, co-transfected luciferase vector and miR-490-3p mimic or miR-490-3p mimic NC into cells. The cells were transplanted into 96-well plate and cultured for 48 h. Eventually, lysed the cells and detected the luciferase activity by dual-luciferase report analysis system (Promega, Madison, WI, USA).
7
Validating circNFIX and CCNB1 Binding to miR-34a-5p
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The binding sites between circNFIX or CCNB1 and miR-34a-5p were predicted by the biological prediction websites miRanda and RNAhybrid, respectively. In the dual-luciferase reporter assay, the corresponding circNFIX and CCNB1 wild-type and mutant sequences of miR-34a-5p were constructed and fused with the luciferase vector pmiR-RB-Report™ (Ribobio, Guangzhou, China). The wild-type and mutant luciferase reporter plasmids of circNFIX and CCNB1 were co-transfected with miR-34a-5p mimics and mimic NC, respectively, into HEK-293T cells. After 48 h of transfection, the cells were harvested and lysed, and luciferase activity was detected using a dual-luciferase report analysis system (Promega, USA).
8
Validating circNFIX and CCNB1 Binding to miR-34a-5p
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The binding sites between circNFIX or CCNB1 and miR-34a-5p were predicted by the biological prediction websites miRanda and RNAhybrid, respectively. In the dual-luciferase reporter assay, the corresponding circNFIX and CCNB1 wild-type and mutant sequences of miR-34a-5p were constructed and fused with the luciferase vector pmiR-RB-Report™ (Ribobio, Guangzhou, China). The wild-type and mutant luciferase reporter plasmids of circNFIX and CCNB1 were co-transfected with miR-34a-5p mimics and mimic NC, respectively, into HEK-293T cells. After 48 h of transfection, the cells were harvested and lysed, and luciferase activity was detected using a dual-luciferase report analysis system (Promega, USA).
9
Validating miR-150-5p Targeting of BTG2
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The targeting relationship between miR-150-5p and BTG2 was predicted through the bioinformatic website Starbase. Mutant (MUT) BTG2 was used to compare the uorescence intensity with wild-type (WT) BTG2 that bound with miR-150-5p, validating that BTG2 was targeted by miR-150-5p. For the 3'untranslated region (UTR) assay, 293T cells with stably expressed miR-150-5p were cultured in 96-well plates and co-transfected with 50 ng BTG2-3'-UTR-WT or BTG2-3'UTR-MUT and control luciferase reporter gene (10 ng pRLCMV renilla luciferase reporter gene) by Lipofectamine 2000 (Thermo Fisher Scienti c Inc). After 48 hours, cell lysates were collected. The re ies and renilla luciferase activity was measured through the dual luciferase report analysis system (Promega, Madison, WI, USA) [20] .
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