GES-1 cells were plated on 24-well plates at a density of 5×104 cells/well the day prior to transfection. The CDX2 or MUC2 promoter luciferase constructs or the pNF-κB-Luc plasmid were co-transfected with a pRL-TK construct (Promega Corporation) into the cells using Lipofectamine® 2000. A total of 24 h post-transfection, cells were incubated in medium containing 200 µmol/l bile acids for an additional 48 h and were harvested for analysis with the Dual-Luciferase Reporter Assay system (Promega Corporation), according to the manufacturer's protocol. Luciferase activity was measured using a PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer, Inc., Waltham, MA, USA). Luciferase intensity was normalized to Renilla luciferase activity to normalize for transfection efficiency. Each experiment was repeated three times.
Prl tk construct
Manufactured by Promega
Sourced in United States
The PRL-TK construct is a laboratory tool used in molecular biology research. It contains a thymidine kinase (TK) gene coupled with a prolactin (PRL) promoter. This construct can be used to study gene expression and regulation, but a detailed description of its intended use or applications is not available.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Enspire 2300 multilabel reader, by PerkinElmer (1 mentions)
Pik3ca, by Takara Bio (1 mentions)
Mutanbest kit, by Takara Bio (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
2 protocols using prl tk construct
1
Bile Acids Regulate Transcription Factors
2
Regulation of PIK3CA by miR-142-5p
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The 3'-UTR fragment of PIK3CA (Genbank accession no. NM_006218.3) containing the putative binding sequence was amplified using the primers 5'-AAAGATAACTGAGAAAATGAAAGCTC-3' (forward) and 5'-GAAGAAAGCTGACCATGCTGCTATG -3' (reverse). The resulting PCR product was cloned into a firefly luciferase reporter vector (pGL3; Promega Corporation, Madison, WI, USA), and termed pGL3-PIK3CA-3'UTR. A plasmid that carried mutations in the complementary sites for the seed region of miR-142-5p was generated based on the pGL3-PIK3CA-3'UTR plasmid using a MutanBEST Kit (Takara Bio Inc., Shiga, JP), and termed pGL3-PIK3CA-3'UTR-mut. The correctness of the plasmids was confirmed by sequence analysis.
HEK293T cells were co-transfected pGL3-PIK3CA-3'UTR or pGL3-PIK3CA-3'UTR-mut and the miR-142-5p mimic or NC duplex. The cells were collected 48 hrs after transfection, and were analyzed for luciferase activity using the dual-luciferase reporter assay kit (Promega Corporation). The pRL-TK construct (Promega Corporation, Madison, WI, USA) was also transfected as a normalization control.
HEK293T cells were co-transfected pGL3-PIK3CA-3'UTR or pGL3-PIK3CA-3'UTR-mut and the miR-142-5p mimic or NC duplex. The cells were collected 48 hrs after transfection, and were analyzed for luciferase activity using the dual-luciferase reporter assay kit (Promega Corporation). The pRL-TK construct (Promega Corporation, Madison, WI, USA) was also transfected as a normalization control.
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