We generated and cloned a sequence including the putative binding site of LINC00847 and the 3′UTR of STK31 as well as the corresponding mutant sequence (GenePharma, Shanghai, China) into the pGL3 luciferase reporter vector. Lipofectamine 2000 (11668019, Invitrogen, USA) was utilized for all co-transfection assays. The co-transfection of BxPC-3 and PANC-1 cells with transcription plasmids and either a microRNA-543 mimic or a control mimic was carried out. At 24 hours after transfection, evaluation of relative luciferase activity was performed using the Nano-Glo® Dual-Luciferase Reporter kit (E1910, Promega, USA) in accordance with the manufacturer’s instructions.
Nano glo dual luciferase reporter kit
Manufactured by Promega
Sourced in United States
The Nano-Glo® Dual-Luciferase Reporter kit is a laboratory equipment product that allows for the simultaneous detection and quantification of two different luciferase reporter proteins within the same sample. The kit includes the necessary reagents and components to perform this dual-luciferase assay.
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Lab products found in correlation
2 protocols using nano glo dual luciferase reporter kit
1
Luciferase Assay of LINC00847 and STK31
2
Validating the circ_0009096-miR-370-3p Interaction
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The association of hsa_circ_0009096 with hsa-miR-370-3p in 293T cells was validated through the dual luciferase reporter assay using the Nano-Glo Dual-Luciferase® Reporter kit (#N1630; Promega, Madison, WI, USA) following the manufacturer’s instructions. Briefly, the circ_0009096 wild-type or mutant sequences were, respectively, ligated with the pmirGLO vectors, which were induced into 293T cells using the Lipofectamine 3000 kit as introduced above, along with the hsa-miR-370-3p mimics (5′-GCCUGCUGGGGUGGAACCUGGU-3′) or its negative control sequences (5′-UUCUCCGAACGUGUCACGUTT-3′). Finally, the cultured 293T cells were lysed 48 h after transfection, and cell lysates were used to measure luciferase activities using a GloMax luminometer. The binding of circ_0009096 with hsa-miR-370-3p was assessed by alterations of luciferase activities between different groups.
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