The synthetized 3′UTR of RUNX2 cDNA containing miR‐149 binding site and mutation site were cloned into pGL3 vector (Promega, Madison, WI) to generate pGL3‐RUNX2‐wild type (Wt) and pGL3‐RUNX2‐mutant type (Mut) plasmids, respectively. HEK293T cells were seeded into a 24‐well plate at 2 × 105 cells/well and cultured for 24 h. Next, 200 ng pGL3‐RUNX2‐Wt or pGL3‐RUNX2‐Mut plasmid was co‐transfected into 293 T cells with miR‐149 mimic or mimic control using Lipofectamine 2000 (Invitrogen). The dual‐luciferase reporter gene analysis system (Promega) was then used to assess luciferase activity. Firefly luciferase activity was standardized to renilla luciferase activity.
Dual luciferase reporter gene analysis system
Manufactured by Promega
Sourced in United States, China
The Dual-luciferase reporter gene analysis system is a tool used to measure the activity of two different luciferase reporter genes simultaneously. It provides a method for rapid and quantitative analysis of gene expression and regulation in living cells.
Automatically generated - may contain errors
Lab products found in correlation
K801 200, by Abcam (7 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (3 mentions)
Pgl3 vector, by Promega (2 mentions)
Luciferase detection kit, by Promega (2 mentions)
Dual luciferase reporter gene plasmid vector, by RiboBio (2 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase detection kit, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Prl tk, by Takara Bio (1 mentions)
Dual luciferase assay, by Promega (1 mentions)
Pgl2 base vector, by Promega (1 mentions)
Luciferase assay kit, by Abcam (1 mentions)
Mir 21 mimic, by RiboBio (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Prl tk, by Promega (1 mentions)
Pmirglo, by Promega (1 mentions)
37 protocols using dual luciferase reporter gene analysis system
1
Regulation of RUNX2 by miR-149
2
Luciferase Assay for LINC00176 Target
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the bioinformatics analysis, target genes of LINC00176 were predicted. The promoter region of CP was constructed onto the pGL3‐Basic vector (Promega Corp.) to produce pGL3‐CP recombinant vector. The correctly sequenced luciferase reporter plasmid was successfully acquired and cotransfected with oe‐LINC00176, oe‐NC, si‐LINC00176 and si‐NC into HEK293T cells, which were then collected and lysed 48 hours later. The luciferase activity was measured using a dual‐luciferase detection kit (K801‐200, Biovision Inc) in a dual‐luciferase reporter gene analysis system (Promega Corp.). Renilla luciferase served as an internal reference. The relative activity of luciferase was indicative of the activity degree of target reporter gene, which equalled the ratio of relative light unit (RLU) of firefly luciferase to RLU of Renilla luciferase.
3
Validation of Circular RNA Binding Sites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The binding sites of circ-RANGAP1 or MYLIP 3′-UTR and miR-542-3p were predicted on the website (https://cm.jefferson.edu/rna22/Precomputed/ ). The miR-542-3p binding site mutation vector of pGL3-RANGAP1-3′-UTR was completed by GeneCreate (Wuhan, China). The wild-type plasmid containing the target sequence was named RANGAP1-WT. The site-directed mutant plasmid was RANGAP1-MUT. MYLIP 3′-UTR promoter sequence containing miR-542-3p binding sites were synthesized to construct MYLIP 3′-UTR wild-type plasmid (MYLIP-WT), and MYLIP3′-UTR mutant plasmid (MYLIP-MUT) was constructed based on site-directed mutation. The MYLIP-MUT and MYLIP-WT were produced by GeneCreate. MG63 cells were transfected, thereafter to measure luciferase activity in the dual-luciferase reporter gene analysis system (Promega, USA) [14 ].
4
Dual-Luciferase Assay for miRNA Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IGF2BP2-3′ UTR and lncRNA MALAT1 cDNA fragments with the miR-204 binding site were inserted into the pGL3 plasmids. More specifically, IGF2BP2-3′ UTR-mut and lncRNA MALAT1-mut fragments were constructed by site-directed mutagenesis and subsequently inserted into pGL3 plasmids with the insertion confirmed by sequencing. Lipofectamine 2000 kits were employed to co-transfect recombinant vectors of pGL3-MALAT1, pGL3-MALAT1-mut, pGL3-IGF2BP2-3′ UTR, and pGL3-IGF2BP2-3′ UTR-mut with miR-204 mimic or NC-mimic into HEK293T cells, respectively. Following a 48-h period of transfection, the cells were harvested and lysed. The relative light unit (RLU) was detected using luciferase assay detection kits (K801-200; BioVision, Milpitas, CA, USA) with Renilla luciferase activity serving as the internal reference. Luciferase reporter gene detection was performed using the dual-luciferase reporter gene analysis system (Promega, Madison, WI, USA). The relative luciferase activity was expressed as the ratio of the RLU value of Firefly luciferase to the RLU value of Renilla luciferase.
5
Luciferase Assay for miR-144 Targeting KLF2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The KLF2 3′ untranslated region (3′UTR) fragment, containing binding sites to miR‐144, was inserted into the pGL3 plasmid. By using the point mutation method, the KLF2‐3′UTR‐mutant (MUT) fragment containing mutated binding sites was constructed and inserted into the pGL3 plasmid. Through liposome transfection, the correctly sequenced pGL3‐KLF2‐wild‐type (WT) and pGL3‐KLF2‐MUT recombinant vector were cotransfected into HEK293T cells with either miR‐144 mimic or NC mimic. The cells were collected and lysed after being transfected for 48 hours, and the relative light unit of each sample was detected using a luciferase detection kit (K801‐200, Biovision) at dual‐luciferase reporter gene analysis system (Promega). The relative luminescence activity was determined as the ratio of the firefly luciferase relative light unit (RLU) to the Renilla luciferase RLU (internal reference).
6
Investigating miR-27 Regulation of NEDD4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The binding site fragments between miR-27 and NEDD4 mRNA 3′-untranslated region (UTR) and mutant fragments were inserted into the luciferase reporter vector Pmir-GLO Dual-Luciferase miRNA Target Expression Vectors (Promega, Madison, WI, USA), namely, reporter plasmids NEDD4-wild type (WT) and NEDD4-mutant type (MUT). NEDD4 luciferase reporter plasmids were co-transfected with mimic NC and miR-27 mimic into 293T cells (Oulu Biotechnology). Luciferase reporter gene detection was performed using a dual-luciferase reporter gene analysis system (Promega). Renilla luciferase was used as an internal reference, and the activation degree of the target reporter gene was compared based on the ratio of the firefly luciferase activity to Renilla luciferase activity.
7
Regulation of KCNQ1OT1 and NLRP3 by miR-30e-3p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The targeting relationship between miR-30e-3p and KCNQ1OT1 or NLRP3 3ʹUTR was examined by luciferase reporter gene assay. The wild type (WT) reporters (KCNQ1OT1-WT or NLRP3-WT) and mutant type (MUT) reporters (KCNQ1OT1-MUT or NLRP3-MUT) containing the predicted miR-30e-3p binding sites were subcloned to pmirGLO dual-luciferase miRNA target expression vector (Promega Corp., Madison, WI, USA). Subsequently, they were co-transfected into HMC3 cells with miR-30e-3p mimics or NC mimics, respectively. After 48 h of transfection, the luciferase activity was assessed by the dual-luciferase reporter gene analysis system (Promega, Madison, WI, USA).
8
CircRNA Interactome Analysis Methods
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
circRNA in vivo precipitation (circRIP), RIP experiments, and luciferase reporter assays were performed as described [38 (link)]. For circRIP and RIP experiments, the A549 cells were cotransfected with the corresponding plasmids and miRNA mimics using Lipofectamine 2000. After treatment, the harvested cells were directly lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China) with protease and RNase inhibitors. Then, the clear supernatant was incubated with the corresponding probes for coprecipitation at 4°C overnight. After treatment with proteinase K, the immunoprecipitated RNAs were extracted by a RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer’s instructions. Additionally, the luciferase activity was examined using the dual-luciferase reporter gene analysis system (Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions.
9
Regulation of TUG1 by SP1 and miR-421
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the analysis results from UCSC (http://genome.ucsc.edu/ ) and JASPAR (http://jaspar.genereg.net/ ), binding sites between SP1 protein and TUG1 were gained. Recombinant luciferase reporter gene vector was co-transfected with oe-SP1 into Hs 675.T, DiFi cells. Renilla luciferase expression vector pRL-TK (TaKaRa, Dalian, China) was utilized as an internal control. Subsequently, the luciferase activity was detected using a dual-luciferase detection kit (K801-200, Biovision) in a dual luciferase reporter gene analysis system (Promega, Madison, WI, USA).
TUG1 3’UTR or KDM2A wild-type (WT) or mutant (MUT) sequences were synthesized and cloned into the pMIR-reporter in the light of the luciferase detection kit (Promega). Using Lipofectamine 2000 reagent, Hs 675.T cells were co-transfected with WT or MUT plasmids and miR-421 mimic or mimic NC. After 48-h of transfection, the cells were harvested for luciferase activity detection by means of a dual luciferase assay (Promega).
TUG1 3’UTR or KDM2A wild-type (WT) or mutant (MUT) sequences were synthesized and cloned into the pMIR-reporter in the light of the luciferase detection kit (Promega). Using Lipofectamine 2000 reagent, Hs 675.T cells were co-transfected with WT or MUT plasmids and miR-421 mimic or mimic NC. After 48-h of transfection, the cells were harvested for luciferase activity detection by means of a dual luciferase assay (Promega).
10
Validating ORC6-miR-1-3p Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The binding sequence between ORC6 and miR-1-3p was co-predicted with miRmap, microT, and miRanda. Accordingly, wild type ORC6 3′UTR sequence (ORC6 WT) and mutant type ORC6 3′UTR sequence (ORC6 Mut) were sub-cloned into pmirGLO dual-luciferase miRNA target expression vector (Promega Corp., Madison, WI, United States), and then, respectively, transfected into Huh7 cells, together with miR-1-3p mimics or control miRNAs. Thirty-six hours later, the luciferase activity was examined by a dual-luciferase reporter gene analysis system (Promega, Madison, WI, United States).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!