Binding sites between miR-140-5p and WNT1 were first predicted on the Bioinformatics & Research Computing website (www.targetscan.org) and the fragment sequence containing the binding sites was obtained. Synthetic WNT1-WT or WNT1-MUT was inserted into the 3ʹUTR of the pMIR-reporter (Thermo Fisher Scientific, Waltham, USA) to package recombinant plasmids pMIR-WNT1-WT and pMIR-WNT1-MUT, respectively. Correctly identified recombinant plasmids WNT1-WT or WNT1-MUT were co-transfected into HEK293T cells with miR-140-5p or NC plasmids, respectively. After incubating for 48 h, cells were cleaved in 1 × passive lysate, and luciferase activity was assessed using a luciferase test kit (Promega, Madison, USA) using a dual-luciferase reporter assay system (Promega). The related target effect was shown as relative luciferase activity (the ratio of firefly luciferase intensity to that of renilla). Renilla luciferase activity was used as the internal reference.
Luciferase test kit
Manufactured by Promega
Sourced in United States
The Luciferase test kit is a laboratory reagent designed to detect and quantify the presence of the luciferase enzyme. Luciferase is a naturally occurring bioluminescent protein that catalyzes a light-producing chemical reaction. The kit provides the necessary components to perform this assay, enabling researchers to measure luciferase activity in various experimental systems.
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Lab products found in correlation
2 protocols using luciferase test kit
1
Validation of miR-140-5p Binding to WNT1
2
Verifying let-7d Targeting of KDM3A
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The targeting correlation between let‐7d and KDM3A was verified using online prediction software and the dual luciferase reporter gene assay. The binding site between let‐7d and KDM3A was analysed to obtain the fragment sequence containing the action site. The 3′‐untranslated region (3′‐UTR) sequence of KDM3A with wild type (WT) or mutated (MUT) let‐7d binding site was cloned into the target sequence of psiCheck2 plasmid downstream of luciferase reporter gene (KDM3A‐WT and KDM3A‐MUT). Subsequently, the luciferase report vectors were co‐transfected into cells with NC mimic, let‐7d mimic, inhibitor‐NC and let‐7d inhibitor, and the resultant luciferase activity was measured using the luciferase test kit (Promega). Following incubation for 48 h, the cells were split in 1 × passive cleavage, and the firefly luciferase activity was measured employing the Dual Luciferase Reporter Assay System (Promega) with renilla luciferases as internal reference.
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