Luminometer td 20 20 detector

The target genes for let‐7a‐1, let‐7d, and let‐7f‐1 were predicted at https://cm.jefferson.edu/rna22/Interactive/, and whether STAT3 was a target gene of let‐7a‐1, let‐7d, and let‐7f‐1 was further validated by dual‐luciferase reporter gene assay. The 3’UTR fragments of STAT3 were artificially synthesized and inserted into pGL3‐control (Promega), that was pGL3‐STAT3‐wild type (WT). The mutations of the 3’UTR were generated, after which the mutant sequence was inserted into the pGL3‐control, referred to as pGL3‐STAT3–mutant type (MUT). The two aforementioned plasmids were transfected with let‐7a‐1 mimic, let‐7d mimic, or let‐7f‐1 mimic into HEK‐293T cells, respectively (Shanghai Institutes for Biological Sciences, CAS, Shanghai, China). Meanwhile, the Renilla luciferase expressing vector pRL‐TK was cotransfected as the internal control for the detection of luciferase activity. The cells were then lysed after 48 hours. The luciferase activity was subsequently detected in the LuminometerTD‐20/20 detector (E5311, Promega) using the Dual‐Luciferase Reporter Assay System Kit (Promega).

The biological prediction website microRNA.org was employed to analyze the target genes of miR-155, and dual-luciferase reporter gene assay was employed to verify that the candidate SOCS1 was a direct target gene of miR-155. The SOCS1-3’-untranslated region (UTR) sequence and the mutated SOCS1-3’-UTR sequence were synthesized. The two synthesized target gene fragments were cloned into the pmir GLO dual-luciferase reporter gene vector to construct the SOCS1-3’-UTR dual-luciferase reporter wild-type gene vector (pmir GLO-SOCS1) and its mutant vector (pmir GLO-mut-SOCS1). The recombinant vector was investigated by polymerase chain reaction (PCR), electrophoresis, and gene sequencing to prove that it had been successfully constructed. HEK-293T cells (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) were respectively co-transfected with the two recombinant vectors and miR-155 mimic or miR-155 NC by using the transfection reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48 hours of transfection, cells were lysed. The luciferase activity was detected on a Luminometer TD-20/20 detector (E5311, Promega, Madison, WI, USA) using a Dual-Luciferase^® Reporter Assay System kit (Promega, Madison, WI, USA) (Song et al., 2017 (link)).

After restriction endonuclease digestion, T4 DNA ligase was used to insert the target fragment of the miR-375 gene promoter into the pGL3-basic reporter plasmid. Next, the WT and MUT luciferase reporter plasmids with correct sequences were cotransfected with oe-NC or oe-WT1 into HEK-293T cells (Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai Research Center of Life Sciences, Shanghai, China). The SIX4 3'UTR gene fragment was artificially synthesized and introduced into the pMIR-REPORT vector (Promega, USA) using HindIII and BamHI endonucleases. The complementary mutation site of the seed sequence was introduced into the wild type SIX4 sequence. After restriction endonuclease digestion, T4 DNA ligase was used again to insert the target fragment into the pMIR-REPORT vector. After that, the WT and MUT luciferase reporter plasmids with correct sequences were cotransfected with miR-375 mimic into HEK-293T cells for 48 h. After cell lysis, the luciferase activity was detected using a Luminometer TD-20/20 detector (model: E5311, Promega, USA) according to the instructions of the Dual-Luciferase Reporter Assay System kit (Promega, USA). The experiment was repeated three times to obtain the mean value.

The artificially synthesized gene fragments Myogenin, MCK, and SV40 were introduced into the Myogenin-Luc, MCK-Luc, and SV40-Luc report plasmids (Promega Corporation, Madison, WI, USA). The luciferase reporter plasmids were co-transfected with LV-MALAT1-shRNA, LV-MyoD-vector, LV-MALAT1-vector, and LV-MyoD-vector into cultured VSMCs. After 48 h of transfection, the VSMCs were collected and lysed, and the luciferase activity was detected with a Luminometer TD-20/20 detector (E5311, Promega Corporation, Madison, WI, USA) using a dual-luciferase reporter assay system kit (Promega Corporation, Madison, WI, USA). The experiment was repeated three times in each group to obtain the mean value.

The synthetic Nox1 3’untranslated region (UTR) gene fragment was introduced into the pGL3-reporter (Promega, Madison, WI, USA) using the endonuclease sites XhoI and BamH I. Then, the complementary sequence mutation site of the seed sequence was designed based on the Nox1 wild type (WT). The digested sequence was inserted into the pGL3-reporter vector using T4 DNA ligase. The constructed luciferase reporter plasmid Nox1 WT or mutant (MUT) was co-transfected into HEK293T cells with miR-298-5p mimic or NC-mimic, respectively. The cells were harvested and lysed 48 h after transfection. Luciferase activity of cells was determined using the Luminometer TD-20/20 detector (E5311, Promega, Madison, WI, USA) on the Dual Luciferase^® Reporter Assay System (E1910, Promega, Madison, WI, USA).

As per the binding sequence between CK2α mRNA 3′ untranslated region (UTR) and miR-499, the target sequence and the mutant sequence were designed and synthesized. The artificially synthesized gene fragments of CK2α 3′ UTR and the mutant were introduced into the pGL3 vector (Promega, Madison, WI, USA) for the construction of mutant-type (MUT) plasmids. After transfection for 48 h, the luciferase reporter plasmids wild type (WT) or MUT were co-transfected with miR-499 mimic or NC mimic into HEK293T cells, which were then collected and lysed. Luciferase activity was measured using a Luminometer TD-20/20 detector (E5311, Promega, Madison, WI, USA) and Dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA).

The synthesized Rac1 3′UTR gene fragment was inserted into pGL3 reporter (Promega, WI, USA) by endonuclease sites XhoI and BamHI. Complementary mutation sites of seed sequence were designed on wild-type (WT) Rac1. After restriction endonuclease digestion, the target fragment was inserted into pGL3 reporter vector by T4 DNA ligase. The constructed luciferase reporter plasmid Rac1-WT or mutant (MUT) was cotransfected with miR-93-5p mimic or mimic NC into HEK293T cells. After 48-h transfection, the cells were harvested and lysed. Luciferase activity was detected on Luminometer TD-20/20 detector (E5311, Promega, WI, USA) using dual-luciferase reporter assay system (E1910). The binding relationship between circHIPK3 and miR-93-5p was detected by the same method.