Dual luciferase detection system

The wild type (WT) circ_0000467/EN2 sequence containing the miR-382-5p binding site was amplified and inserted into the empty luciferase reporter vector to obtain the WT-circ_0000467/WT-EN2 reporter vector. The mutant type (MUT) circ_0000467/EN2 sequence was also inserted into the empty luciferase reporter vector to obtain the MUT-circ_0000467/MUT-EN2 reporter vector. The constructed luciferase reporter plasmids were co-transfected with miR-382-5p mimics or miR-control into HEK293T cells planted in a 96-well plate. After 48 h, the activities of Firefly luciferase and Renilla luciferase were determined using a dual-luciferase detection system (Promega, Madison, WI, USA). The relative luciferase activity was normalized to Renilla luciferase activity.

A transient double luciferase reporting (LUC) assay was used to further confirm whether FtbZIP85 promoted the activation or inhibited the transcription of the FtDFR gene. The regulatory region of the FtDFR gene at −2 k bp upstream of ATG was amplified and cloned into the pGreenⅡ0800-LUC (Reporter) vector. The CDS sequence of FtbZIP85 (excluding the stop codon) was amplified and cloned into the pCambia1300-eGFP (Effector) vector. After the successful construction, all vectors were transformed into the Agrobacterium strain GV3101 in the presence of the helper plasmid pSoup-p19. The dye solution formula was: 10 mM MgCl₂/10 mM MES. Agrobacterium-suspensions were mixed separately at a ratio of 1:1 and injected into tobacco leaves. Samples were taken after 2.5 d of continued culture and used for LUC experiments. The dual luciferase detection system (Promega, Madison, WI, USA) was used according to the instructions provided by the evaluation of the ratio between LUC and REN.

To evaluate the function of JMJD2C on MALAT1 promoter activity, A549 cells were transfected with sh-NC or sh-JMJD2C with a luciferase reporter plasmid containing MALAT1 promoter through Lipofectamine 3000 (Invitrogen). After 48 h, cell lysates were subjected to analysis of relative luciferase activity on the dual luciferase reporter gene detection system (Promega, WI, USA) [10 (link)].
The interaction between MALAT1 and miR-503-5p and the targeting relationship between miR-503-5p and SEPT2 were conformed. MALAT1 wild-type (WT), MALAT1 mutant (MUT), SEPT2 WT and SEPT2 MUT potentially binding to miR-503-5p were inserted into pGL4 luciferase reporter and co-transfected with miR-503-5p mimic or mimic-NC into A549 cells. After 48 h, the relative luciferase activity was measured using a dual luciferase detection system (Promega).

The psiCHECK™-2 vector inserts the 3’ UTR of IGF1, which is predicted to bind to miR-222. The vector, miR-222 mimic and negative control were transfected into HeLa cell lines using Lipofectamine 3000 (Invitrogen). 48 h after transfection, cells were collected and lysed, and luciferase activity was measured using a dual-luciferase detection system (Promega, Madison, WI, USA) according to the kit instructions.

The 3ʹUTR of IRF1 was constructed such that it included the conserved binding sites for miR-125-5p and a mutant 3ʹUTR fragment of IRF1 was constructed with the mutations confined to the conserved binding sites for miR-125-5p. The fragments that included the 3ʹUTR regions (IRF1-WT) or mutant 3ʹUTR regions (IRF1-WT) were inserted into vector pcDNA containing a firefly luciferase reporter gene. VSMCs were co-transfected with reporter plasmid and miR-125a-5p mimic or miR-125a-5p inhibitor using Lipofectamine 2000 (Invitrogen) for 48h. Next, the cells were collected, and their luciferase activity was measured using dual Luciferase detection system (Promega) according to the manufacturer’s instructions.