CMPK1 3′-UTR sequence was amplified from cDNA with the CMPK1 3′-UTR up primers SacI (5′-GAGCT′CGCTTCCTTTCATCAGGTATC-3′) and down primers XhoI (5′-CTCGAGCATCCAACATCACTGAATGG-3′). The PCR products were then subcloned to the pmirGLO dual-luciferase target expression vector (Promega, USA) as wild-type vector pmirCMPK1-3′-UTR-Wt (CMPK1-Wt). The mutant vector pmirCMPK1-3′-UTR-Mut (CMPK1-Mut) was obtained by site-directed mutagenesis using QuikChange® Site-Directed Mutagenesis Kit (Stratagene, USA). AGS and MGC-803 were seeded in a 24-well culture plate in triplicate and were co-transfected with miR-130b mimic and miR-NC followed by CMPK1-Wt or CMPK1-Mut using DharmaFECT Duo Transfection Reagent (Thermo, USA) according to the manufacture’s procedure. Luciferase activity was normalized to that of pRL-TK luciferase. The cells were collected at 24h post-transfection; luciferase activity was measured by a dual-luciferase reporter assay kit (Promega, USA) and recorded by a GloMax 20/20 (Promega, USA).
Pmirglo dual luciferase target expression vector
Manufactured by Promega
Sourced in United States
The PmirGLO Dual-Luciferase Target Expression Vector is a tool for the expression and analysis of target genes in mammalian cells. It contains two reporter genes, one for firefly luciferase and one for Renilla luciferase, which can be used to measure the activity of the target gene.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (5 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Glomax 20 20, by Promega (1 mentions)
Dharmafect duo transfection reagent, by Thermo Fisher Scientific (1 mentions)
Plko 1 trc cloning vector, by Addgene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Mir nc, by GenePharma (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Luciferase activity kit, by Promega (1 mentions)
Luciferase reporter vector, by GenePharma (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
11 protocols using pmirglo dual luciferase target expression vector
1
Investigating CMPK1 3'-UTR Regulation by miR-130b
2
Luciferase Assay for miRNA Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amplified sequence of TUG1 or DKK1 including the predicted miR‐34a binding sequence was inserted into a pmirGLO Dual‐luciferase Target Expression Vector (Promega) to produce Wt‐TUG1 or Wt‐TUG1 reporter vector. Dual‐Luciferase Reporter Assay System (Promega) was used to carry out Luciferase reporter assay. The full description of luciferase reporter assay is available in the online data supplements.
3
Validating NEAT1-miR-365 Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fragments of NEAT1 containing wild-type (WT) or mutant type (MT) miR-365 binding sites were cloned into pmirGLO Dual-luciferase Target Expression Vector (Promega Corporation, Madison, WI, USA), which generated WT or MT NEAT1 plasmids. HN4 and Tca-8113 cells were co-transfected with miR-365 mimic, scramble miR mimic (miR-NC), WT or MT NEAT1 plasmid using Lipofectamine 2000. Following 48 h of transfection, luciferase reporter gene assays were performed using the Dual-Luciferase Reporter Assay System (Promega Corporation). The firefly luciferase activity was normalized against Renilla luciferase activity.
4
Functional Validation of OIP5-AS1-miR-26a-3p Axis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The shRNAs targeting OIP5-AS1 were synthesized by Sangon Biotech (Shanghai, China) and inserted into a pLKO.1-TRC cloning vector (Addgene). The target sequence was 5′-GTGACTTAAACAGCTTAAATT-3′ (shRNA1) and 5′-TAAACAGTGACTTTAAATTGT-3′ (shRNA2), respectively. Also, the pLKO.1-TRC control vector was used as a control (named as SCR).
The sequence of the hsa-miR-26a-3p-binding site within the OIP5-AS1 was predicted with starBase 2.0 (http://starbase.sysu.edu.cn ). The fragment of OIP5-AS1 and the EPHA2 3′ UTR containing the binding site of hsa-miR-26a-3p were amplified by PCR and cloned into the pmirGLO Dual-Luciferase target expression vector (Promega, Madison, WI, USA).80 (link) Site mutations were introduced to the WT to construct the MT. All constructions were confirmed by Sanger sequencing.
The sequence of the hsa-miR-26a-3p-binding site within the OIP5-AS1 was predicted with starBase 2.0 (
5
Dual-Luciferase Assay for miRNA-Target Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Firstly, pmirGLO‐MATN1‐AS1‐WT or pmirGLO‐MATN1‐AS1‐mut, pmirGLO‐CHD1‐wt or pmirGLO‐CHD1‐mut was constructed using a pmirGLO Dual‐luciferase Target Expression Vector (Promega, Madison, WI, USA). And miR‐200b/c/429 mimics/inhibitors (for miR‐200b/c/429 overexpression/inhibition, respectively) or miR‐NC was also obtained from RiboBio. Thereafter, these plasmids were appropriately transfected into glioma cells or Hek‐293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) based on the manufacturer's guide. The relative luciferase activity was determined by a dual‐luciferase reporter assay kit (Promega) after 48 hours of transfection.
6
Validating miR-590-3p binding to ZFAS1 and HMGA2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 3ʹUTR sequence of ZFAS1 containing the putative miR-590-3p binding site was amplified using qRT-PCR and cloned into a pmirGLO Dual-luciferase Target Expression Vector (Promega, Madison, WI, USA) to form the reporter vector ZFAS1-wild-type (ZFAS1-Wt). The mutant 3ʹ-UTR sequence of ZFAS1 was designed and the ZFAS1-mutant-type (ZFAS1-Mut) was generated in a similar manner. HEK293T cells were seeded on 24-well plates for 24 h. Afterwards, ZFAS1-Wt and ZFAS1-Mut were transfected into HEK293T cells with miR-590-3p mimics or miR-NC (GenePharma, Shanghai, China) using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, luciferase activity was measured by the Dual-Luciferase Reporter Assay System (Promega). Additional Luciferase reporter assays were performed as described above to determine the direct binding of miR-590-3p to HMGA2 3ʹUTR.
7
Reporter Vector Construction and Luciferase Assay for miR-672-5p Targeting TAB2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 3′-UTR or mutated miR-672-5p binding site of TAB2 subcloned into the pmirGLO Dual-luciferase Target Expression Vector (Promega, Madison, WI, USA) to construct the reporter vector pmirGLO-TAB2-WT and pmirGLO-TAB2-Mut. miR-672-5p mimic or mimic-NC were transfected to BV2 cells, followed by transfection with pmirGLO-TAB2-MT or pmirGLO-TAB2-Mut plasmid. The luciferase activity was determined by luciferase activity kit (promega) according the manufacturer’s protocol.
8
Luciferase Assay for TUG1-MAZ Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The putative TUG1 binding site of the MAZ sequence (MAZ 3'UTR-Wt 5'-GCAGCCAGUGUCCCCCUCCCCUCUU -3') and mutation binding site of MAZ (MAZ 3'UTR-Mut 5'-UGUUGGUGGUAGGGGCGGGGGAGGAA-3') were amplified by PCR and cloned into a pmirGLO Dual-Luciferase Target Expression Vector (Promega, USA) to construct a luciferase reporter vector (GenePharma). HEK-293 T cells were inoculated on 96-well plates, and the cells were cotransfected with MAZ-Wt (or MAZ-Mut) and TUG1 (+) or TUG1(+)-NC plasmids using Lipofectamine 3000. After 48 h, luciferase activity was assessed using a Dual-Luciferase Reporter System.
9
Luciferase Assay for RNA Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The putative LINC00346 binding site of the ZNF655 3′ UTR sequence and the respective putative ZNF655 mRNA binding site of EGFL7, ROBO4, and ANKHD1 mRNA were amplified by PCR and cloned into a pmirGLO Dual-Luciferase Target Expression Vector (Promega, Madison, WI, USA) to construct a luciferase reporter vector (GenePharma). Luciferase assays were performed as previously described.46 (link) Also see Supplemental Materials and Methods for details.
10
Validating miR-21-5p Binding to XIST
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fragment from XIST containing the predicted miR-21-5p binding site was amplified by PCR and cloned into a pmirGLO Dual-luciferase Target Expression Vector (Promega, Madison, WI, USA) to form the reporter vector XIST-wild-type (XIST-Wt). To test the binding specificity, the corresponding mutant was created by mutating the miR-21-5p seed region binding site (seed sequence binding fragment 5′-TATTCGA-3′ changed to 5′-CGTGAGC-3′), which were named as pmirGLO-XIST-Mt (XIST-MUT). pmirGLO-XIST-Mt or pmirGLO-XIST-WT was co-transfected with miR-21-5p mimics, miR-21-5p inhibitor or miR-21-5p NC into HEK 293T cells using Lipofectamine 2000. Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) 48 h later, the firefly luciferase activity was measured and normalized by Renilla luciferase activity.
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!