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.
Pmei and xbai restriction enzymes
Manufactured by New England Biolabs
Sourced in United States
PmeI and XbaI are type II restriction enzymes that recognize and cleave specific DNA sequences. PmeI recognizes the palindromic DNA sequence 5'-GTTTAAAC-3', while XbaI recognizes the palindromic DNA sequence 5'-TCTAGA-3'. These enzymes can be used for a variety of molecular biology applications, such as DNA cloning, genetic engineering, and restriction fragment length polymorphism (RFLP) analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dual glo luciferase assay kit, by Promega (2 mentions)
Victor2 fluorometry, by PerkinElmer (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Noti restriction enzyme, by New England Biolabs (1 mentions)
Jm109 competent cells, by Promega (1 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (1 mentions)
Pmirglo dual luciferase vector, by Promega (1 mentions)
3 protocols using pmei and xbai restriction enzymes
1
Luciferase Reporter Assay for miR-21 Inhibition
2
Generating pmirGLO miRNA Expression Plasmids
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The pmirGLO plasmid construction was based on the manufacturer manuals. The pmirGLO dual-luciferase miRNA target expression vector was purchased from Promega. The vector was digested with PmeI and XbaI restriction enzymes (New England Biolabs). The inserts for pmirGLO miR144 and pmiRGLO miR31 plasmids were purchased from Integrated DNA Technologies (Table S5 ) and were also digested with PmeI and XbaI restriction enzyme. The inserts were each ligated with digested pmirGLO plasmids using a quick ligasation kit (New England Biolabs) and transformed into JM109 competent cells (Promega). The plasmid was verified by digestion with the NotI restriction enzyme (New England Biolabs), according to manual.
3
miRNA Target Validation by Luciferase Assay
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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.
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