First, oligonucleotide pairs containing miR-16, miR-15b, miR-638, miR-515 and miR-194 target regions were ordered and annealed, respectively. Then, five pmirGLO Dual-Luciferase miRNA target expression vectors were constructed by inserting the respective annealed oligonucleotides into the pmirGLO vector (Promega) using PmeI and XbaI restriction enzymes (New England Biolabs, Inc). When fusion rate reached 70%-90%, MKN28 cells were co-transfected with 80 ng of either circNF1 overexpression plasmid (circNF1) or empty vector (EV), along with 80 ng of either pmirGLO vector containing miRNA binding sites or empty pmirGLO vector, into 1.0×104 cells/well using Lipofectamine™ 2000 (Invitrogen). After 24 hours, cells were analyzed for firefly and Renilla luciferase activity using the Dual-Glo® Luciferase Assay Kit (Promega) and VICTOR2 fluorometry (Perkin Elmer, Waltham, MA).
Victor2 fluorometry
Manufactured by PerkinElmer
Sourced in United States
The VICTOR2 is a high-performance fluorometry instrument designed for a wide range of applications. It offers automated, precise, and sensitive detection of fluorescent signals in microplate-based assays. The VICTOR2 is capable of measuring fluorescence intensity, time-resolved fluorescence, and fluorescence polarization.
Automatically generated - may contain errors
Lab products found in correlation
Dual glo luciferase assay kit, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Pmirglo vector, by Promega (2 mentions)
Pmei and xbai restriction enzymes, by New England Biolabs (2 mentions)
Pmirglo vector, by Thermo Fisher Scientific (1 mentions)
Pmirglo dual luciferase vector, by Promega (1 mentions)
7 protocols using victor2 fluorometry
1
Dual-Luciferase Assay of circNF1 and miRNAs
2
Validating circFUT8-miR-944 Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The possible target of circFUT8 and miR-944 were shown by starBase v2.0 (http://starbase.sysu.edu.cn/
3
Circulating RNA-miRNA Interaction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Based on bioinformatics prediction starbase (http://starbase.sysu.edu.cn/ ), Circbank (http://www.circbank.cn/ ), and Circular RNA interactome (https://circinteractome.irp.nia.nih.gov/ ), miR-625-5p was selected as a candidate target of circMMP11. The complementary sequences between miR-625-5p and ZEB2 were presented by Targetscan (http://www.targetscan.org/vert_72/ ). Partial sequences of circMMP11 and 3′UTR of ZEB2 that contain the putative binding sites for miR-625-5p were synthesized and cloned into luciferase report vectors (Ambion, Foster City, CA, USA). Subsequently, the BC cells were co-transfected with miR-625-5p mimic or miR-NC and the wild or mutant type reports. The relative firefly luciferase activity was determined under the VICTOR2 fluorometry (PerkinElmer, Waltham, MA, USA) and normalized to Renilla luciferase activity.
4
Luciferase Reporter Assay for miR205HG-PTCH1 Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confluent cultures of miR205HG-stable cells were plated onto six-well plates at 1.5 × 105 cells/well. On the next day, we cotransfected cells with Renilla luciferase and either pmirGLO-PTCH1 or pGL3-8xGLI (gifted by Dr. Philip Beachy, Stanford University, Stanford, CA, USA; the 8X Gli fragment was subcloned into the pGL3 promoter vector) using BioT transfection reagent (2 μL well) according to manufacturer’s protocol. After cells reached saturation density (one–two days), they were changed to low-serum medium (0.5% bovine calf serum). Cells were lysed and transferred to 96-well plates prior to luciferase reporter assays using Dual-Glo luciferase assay kits (Promega, Madison, WI, USA). Luminescence intensity was measured by VICTOR2 fluorometry (Perkin Elmer, Waltham, MA, USA), and luminescence intensity of Firefly luciferase was normalized to that of Renilla luciferase.
5
Luciferase Reporter Assay for miR-21 Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A luciferase reporter vector containing the miR-21 binding site was constructed by inserting annealed miR-21 binding site (IDT) into the pmirGLO vector (Promega, Madison, WI, USA), using PmeI and XbaI restriction enzymes (New England Biolabs). At 70%–80% confluence, NCI-N87, AGS, and MKN28, which express high endogenous levels of miR-21 based on our qRT-PCR results (described in Results ), were co-transfected using Lipofectamine 2000 (Invitrogen) with 15 nM of either negative control scrambled circRNA or miR-21 inhibitor (MH10206; Thermo Fisher Scientific) or scRNA21, together with 80 ng of either luciferase reporter plasmid containing the miR-21 binding site, or empty pmirGLO vector per well of a 96-well plate. 24 hr after transfection, cells were analyzed for luciferase activity using a Dual-Glo Luciferase Assay Kit (Promega) and VICTOR2 fluorometry (Perkin Elmer, Waltham, MA, USA). Firefly luciferase activity was normalized to that of Renilla luciferase. All transfections were performed in triplicate, whereas luciferase activity was averaged from duplicate experiments.
6
Validating circ-UBR4 and miR-515-5p Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The downstream targets of circ-UBR4 and miR-515-5p were predicted by circRNA interactome (https://circinteractome.irp.nia.nih.gov/ ) and Starbase (http://starbase.sysu.edu.cn/ ), respectively. The circ-UBR4 sequences containing miR-515-5p binding sites were inserted into pmirGLO luciferase vector (GeneCreat, Wuhan, China), named as WT-circ-UBR4, with MUT-circ-UBR4 as control. Similarly, the wild- and mutant-type of IGF2 luciferase reporter vector (WT-IGF2 3′UTR and MUT-IGF2 3′UTR) were synthesized by GeneCreat. VSMCs were transfected with luciferase reporter vectors in the presence or absence of miR-515-5p by Lipofectamine 2000 (Thermo Fisher Scientific). Relative luciferase activity was assessed under the VICTOR2 fluorometry (PerkinElmer, Waltham, MA, USA) at 48 h post-transfection.
7
miRNA Target Validation by Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First, we successfully constructed pmirGLO Dual-Luciferase vector (Promega, Madison, WI) containing miR-21 or miR-93 target sequences by inserting either miR-21 or miR-93 binding sites using PmeI and XbaI restriction enzymes (New England Biolabs). Next, 1.0 × 10 4 cells/well of SKGT4 and OE33 were seeded in 96-well plates and cotransfected with different concentrations (2, 3, 5 nM) of either circ-21-93 or circ-scr and 80 ng pmirGLO miR-21 / miR-93 target site vector. 24 h after transfection, cells were analyzed for Firefly luciferase activity using the Dual-Glo ® Luciferase Assay Kit (Promega) and VICTOR2 fluorometry (Perkin Elmer, Waltham, MA) and normalized to 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!