Circbank online database was performed to assess the targeting sequence of miR-708-5p in circ_0009112. The wide-type (wt) sequence of circ_0009112 was amplified and sun-cloned into pmirGLO vector (Promega, Madison, WI, United States), and named as wt-circ_0009112. The mutant (mut) circ_0009112 without the binding sites of miR-708-5p in circ_0009112 was synthesized and inserted into pmirGLO vector (Promega), and named as mut-circ_0009112. The built plasmids were transfected into SaOS2 and U2OS cells with miR-708-5p mimic or miR-NC, respectively, with Lipofectamine 2000 (Thermo Fisher). Luciferase activities were determined by luciferase activity detection kit (Promega). Ranilla Luciferase activity served as a control of firefly luciferase activity.
Luciferase activity detection kit
Manufactured by Promega
Sourced in United States
The Luciferase Activity Detection Kit is a laboratory tool designed to measure the activity of luciferase, a bioluminescent protein. The kit provides reagents and protocols to quantify luciferase expression in cell-based assays, allowing researchers to study gene expression, cell signaling, and other biological processes.
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5 protocols using luciferase activity detection kit
1
Assessing miR-708-5p Regulation of circ_0009112
2
Luciferase Assay of miR-200b Regulation
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The CAF-co-cultured cells were inoculated into 96-well plates at 3×104 cells/well. After culturing for 24 hours, luciferase reporter vectors (HMGB3-MT and HMGB3-WT) and miR-200b mimics were co-transfected into the cells. After transfection for 48 hours, the luciferase activity was examined according to the luciferase activity detection kit instructions (Promega, Madison, WI, USA). The luciferase activity = firefly luciferase activity value/renilla luciferase activity value.
3
Investigating miR-361-3p and Wnt10A Interactions
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The relationship between miR-361-3p and Wnt10A is estimated by https://www.ncbi.nlm.nih.gov/nuccore/NM_025216
4
Validating PLXNA1-miR-134 Interaction
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The relationship between PLXNA1 and miR-134 was predicted using MicroRNA.org , a biological prediction website. The 3′ untranslated region (UTR) sequence of PLXNA1 was synthesized and then inserted into pMIR-reporter (Promega, Madison, WI, USA). Mutant (Mut) fragment with mutated binding sites of miR-134 in the PLXNA1 3’UTR was also cloned into pMIR-reporter. The pMIR-Wt-PLXNA1 and pMIR-Mut-PLXNA1 were respectively co-transfected with miR-134 into HEK-293 T cells (Shanghai Beinuo Biotechnology Co., Ltd., Shanghai, China). At the 48th h after transfection, the cells were collected and lysed. Then luciferase activity was assessed using the luciferase activity detection kit (Promega).
5
Detecting miR-21-5p Binding to KRIT1 3'UTR
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Wild type (WT) and mutant type (MUT) of KRIT1 3'UTR were inserted into pmirGLO plasmid (between NheI and Sal I) to form luciferase vector. Mimics of miR-21-5p (5'-UAGCUUAUCAGACUGAUGUUGA-3') and negative control (NC) mimics (5'-UUCUCCGAACGUGUCACGUTT-3') were used to detect the bind of miR-21-5p to KIRT1 3'UTR. Transfections int HUVECs were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Luciferase activity of re y was detected with Luciferase Activity Detection Kit (E1910, Promega, USA) using a Chemiluminescence Apparatus (Lumat LB 9507, Berthold, German).
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