MiR-218-5p in circSAMD4A or KLF8 3′-UTR wild-type (WT) and their mutated (MUT) sequences were separately cloned into pRL-TK luciferase plasmids (Promega, Shanghai, China). Then HOS/DXR and U2OS/DXR cells were co-transfected with 100 ng of constructed luciferase reporter plasmid and 40 nM miR-218-5p or miR-NC using Lipofectamine™ 2000 (Invitrogen). Finally, luciferase activity was detected using a dual luciferase assay kit (Promega).
Prl tk luciferase plasmid
Manufactured by Promega
Sourced in China
The PRL-TK luciferase plasmids are a set of reporter gene constructs that contain the luciferase gene from the firefly, Photinus pyralis, under the control of the PRL (Prolactin) promoter and the Herpes Simplex Virus Thymidine Kinase (HSV-TK) promoter. These plasmids are designed to monitor gene expression and transcriptional activity in various cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Prl tk, by Promega (1 mentions)
Passive cell lysis buffer, by Promega (1 mentions)
4 protocols using prl tk luciferase plasmid
1
Dual-Luciferase Assay for miR-218-5p Targets
2
miR-149-5p Binding Site Validation
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The circ_0072995 or SHMT2 3'UTR sequences with wild type (WT) or mutant (MUT) binding sites of miR-149-5p were separately cloned into the pRL-TK luciferase plasmids (Promega, Shanghai, China) to generate pRL-TK-WT-circ_0072995, pRL-TK-MUT-circ_0072995, pRL-TK-SHMT2 3'UTR-WT, or pRL-TK-SHMT2 3'UTR-MUT luciferase reporter, respectively. Then these constructed luciferase reporters with miR-149-5p mimic or miR-NC were transfected into SKBR3 and BT-549 cells using Lipofectamine 2000 (Invitrogen). 48 h later, luciferase activities were examined using a Dual-Luciferase reporter assay kit (Promega).
3
Luciferase Reporter Assay for AP-1, c-Jun, and c-Fos
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JB6 Cl41 and MCF7 cells transfected with AP-1-, c-Jun-, and c-Fos-Luc were lysed in passive cell lysis buffer (Promega). The firefly luciferase activity in cell lysate was measured using the GloMax®-Multi Detection System (Promega). The Renilla luciferase activity generated by pRL-TK-luciferase plasmid (Promega) was used to normalize the transfection efficiency. c-Fos-Luc (pFos-WT GL3) and c-Jun-Luc (JC6GL3) promoter constructs were a gift from Dr. Ron Prywes (Columbia University, NY, USA). AP-1 luciferase reporter plasmid (−73/+63 collagenase-luciferase) was kindly provided by Dr. Dong Zigang (Hormel Institute, University of Minnesota, Austin, MN, USA).
4
AP-1, c-Jun, and c-Fos Luciferase Assay
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To detect firefly luciferase activity, a reporter gene assay was performed using lysates from AP-1-, c-Jun-, and c-Fos-luc-transfected JB6 Cl41 and MCF7 cells. The reporter gene vector pRL-TK-luciferase plasmid (Promega) was co-transfected into each cell line and the Renilla luciferase activity generated by this vector was used to normalize the results for transfection efficiency. Cell lysates were mixed with Luciferase Assay Reagent II, following which firefly luciferase light emission was measured using GloMax®-Multi Detection System (Promega). Subsequently, Renilla luciferase substrate was added to this setup to normalize the firefly luciferase data. The c-Fos-luc (pFos-WT GL3) and c-Jun-luc (JC6GL3) promoter constructs were kindly provided by Dr. Ron Prywes (Columbia University, New York, NY, USA). The AP-1 luciferase reporter plasmid (−73/+63 collagenase-luciferase) was kindly provided by Dr. Dong Zigang (Hormel Institute, University of Minnesota, Austin, MN, USA).
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