The manufactured psiCHECK-2 plasmid was transfected into 293T cell line (ATCC, United States) by Lipo 2000 (Thermo Fisher Scientific, United States), together with the hsa-miR-557 or hsa-miR-557 inhibitor or the negative control (NC), accordingly. Dual-luciferase reporter assays (Beyotime Biotechnology, Shanghai, China) were performed according to the manufacturer’s instruction. Relative light unit (RLU) of firefly and Rinilla luciferase were recorded.
Dual luciferase reporter assay
Manufactured by Beyotime
Sourced in China
The Dual-Luciferase Reporter Assay is a laboratory equipment used to quantify and compare the activity of two different luciferase reporter enzymes simultaneously. It provides a rapid and sensitive method for measuring gene expression and reporter activity in cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pmir report luciferase vector, by RiboBio (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
293t cell line, by American Type Culture Collection (1 mentions)
Lipofectamine 2000 transfection regent, by Thermo Fisher Scientific (1 mentions)
Pmir vector, by Thermo Fisher Scientific (1 mentions)
9 protocols using dual luciferase reporter assay
1
Dual-Luciferase Assay of miR-557
2
Dual-Luciferase Reporter Assay for SNP Function
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Cells were cultured and transfected by using the mixture of 4.2 μg plasmids (including 4 μg firefly luciferase pGL3-basic plasmids and 0.2 μg renilla luciferase pRL-SV40 plasmids) and 3 μL Lipofectamine 3000 (Life, USA) in 12-well plate. Then, the cells were harvest at 24, 48, and 72 h for further studies. Dual-luciferase reporter assays (Beyotime, China) were performed to evaluate the function of two allele of each SNP. The intensities of firefly luciferase and Renilla luciferase (used as controls) were detected at 560 and 465 nm by using Victor X Multimode Plate Reader (PerkinElmer, USA), respectively. Finally, the firefly/Renilla ratio was calculated to analyze the expression levels of firefly.
3
Investigating miR-92b-3p Regulation of PTEN
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Investigating the target genes of miRNAs is crucial to identify their regulatory mechanisms and functions. Here, we identified four common DE-miRNAs and predicted their targets using three miRNA-target tools: miRDB, mirDIP, and TargetScan. The miRNA targets were identified based on overlapping results from the three websites. In this study, we selected phosphatase and tensin homolog (PTEN) as the target gene for further studies. Based on the predicted binding loci for miR-92b-3p in PTEN, luciferase reporter gene plasmids were constructed containing wild-type PTEN (PTEN-wt) and mutant-type PTEN (PTEN-mt) consisting of the PTEN 3′ untranslated region (UTR). The reporter vector, together with the control, experimental miR-92b-3p mimic, or miR-92b-3p inhibitor, was transfected into cells once cells reached 50%–60% confluence. The cells were collected after 48 h to assess the relative activities of firefly and Renilla luciferases using a Dual Luciferase Reporter assay (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer's instructions. All experiments were repeated three times.
4
Validating miR-222-3p Target Gene BRG1
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To validate BRG1 as the target gene of miR-222-3p, a dual-luciferase reporter assay was performed. Briefly, the mutated sequences of the binding sites were designed, and the corresponding vectors were constructed by Shanghai Genechem Co., Ltd. (Shanghai, China). The reporter vector was cotransfected with miR-222-3p mimics into NCM460 cells using Lipofectamine 2000 (Thermo Fisher Scientific, USA). After 48 h, according to the manufacturer’s instructions, the activity of luciferase was detected using a dual-luciferase reporter assay (Beyotime, Shanghai, China).
5
Investigating miR-192-5p Regulation of lncRNA WAC-AS1
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The data base known as starBase was used to identify the relationship between miR-192-5p and lncRNA WAC-AS1. The 3’UTR of lncRNA WAC-AS1, containing the miR-192-5p binding site or mutated target site, was synthesized by carrying out a genomic PCR (ELK Biotechnology) and was cloned into pMIR vectors (Ambion, Austin, TX, USA) to construct the reporter vector lncRNA WAC-AS1 wild-type (lncRNA WAC-AS1-WT) or lncRNA WAC-AS1 mutant-type vector (lncRNA WAC-AS1- MUT). Next, 293T cells were transfected with lncRNA WACAS1- WT or lncRNA WAC-AS1-MUT combined with miR-192-5p mimic or mimic control using Lipofectamine 2000 transfection regent (Invitrogen). The transfected cells were incubated for 48 h and dual-luciferase reporter assay (Beyotime Technology, Jiangsu, China) was performed. Firefly luciferase activity of the plasmids was normalized to Renilla luciferase activity.
6
Validating miR-145 Target FOXO1
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According to the bioinformatics analysis, we predicted the potential target genes for miR-145. Forkhead box O1 (FOXO1) was found to have the complementary sequence of miR-145 at the 3′-UTR. To verify the target gene, a luciferase activity assay was performed. First, the wild-type (WT) and the mutant-type (MUT) 3′-UTR of FOXO1 were separately combined into the pMIR-Report luciferase vector (RiboBio, Guangzhou, China). Second, the VECs were co-transfected with the recombined pMIR report plasmid or the pMIR-control and miR-145 mimic or miR-NC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the protocols of manufacturers. The luciferase activity under different treatments was examined by a Dual-Luciferase Reporter Assay (Beyotime, Jiangsu, China).
7
Characterizing miRNA-371a-5p Binding
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COLCA1 full-length and SPP1 3’-UTR of the wild-type (WT) and mutant-type (Mut) hsa-miR-371a-5p binding site was generated by Guangzhou All-perfect Biological Technology Co., Ltd. (Guangzhou, China). COLCA1-WT/Mut or SPP1-WT/Mut was transfected into 293T cells with hsa-miR-371a-5p mimics/NC mimics or hsa-miR-371-5p inhibitor/NC inhibitor. Luciferase activity was evaluated by the Dual-Luciferase Reporter Assay (Beyotime Biotechnology, China) after 48 hours of transfection.
8
Luciferase Assay for miR-1207-5p Targeting
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To generate the pMIR‐RB‐3′‐UTR construct, the STAT6 3′‐UTR containing the target sites of miR‐1207‐5p and its mutant form were cloned into the pMIR‐Report luciferase vector (Guangzhou RiboBio) between the HindIII and SacI sites. Approximately 1 × 105 human breast cancer cells were seeded in 12‐well plates and cultured for 24 h. Then 2 μg pMIR‐control or recombinant pMIR report plasmid and 50 nM miR‐1207‐5p mimic or negative control were cotransfected into cells. The activity of firefly luciferase was measured using the Dual‐Luciferase Reporter Assay (Beyotime, Jiangsu, China). The relative luciferase activity was normalized to the firefly luciferase activity.
9
Validating HMGN2 3' UTR as miR-155/23a Target
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For experimental validation of the HMGN2 3′ UTR as a target of miR-155 or miR-23a, co-transfections of reporter constructs and miR-155 (or miR-23a) mimic were carried out in A549 cell. After 24 hours of transfection, cells were lysed and luciferase activity was measured on 96-well black plates in a Microplate reader (Thermo, USA). Luciferase activities were measured by the relative activity of Renilla/firefly luciferase unit (RLU) using a Dual-Luciferase Reporter Assay (Beyotime Institute of Biotechnology, Haimen, China).
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