The 3’ untranslated region (UTR) of RSK2, S6K1 and PDGFRα were cloned into pMIR-REPORT miRNA expression reporter vector (ABI). Twenty four hrs after transfection with negative or miR-218 mimics in T98G cells, the respective plasmids were transfected into cells with Fugene 6 transfection reagent (Roche) in 24-well plates. Renilla Luciferase plasmid was co-transfected and utilized to normalize the firefly Luciferase values expressed from the pMIR-REPORT expression vector. Luciferase assays were performed using the dual luciferase protocol (Promega) at 48 hrs post transfection of the corresponding plasmids containing the 3’UTR regions. For testing the role of specific seed sequences in the 3’UTR of miR-218 target genes, mutations were introduced into the miR-218 binding seed sequences and compared to the wild type sequence (table S2 ). The duplex sequences spanning the miR-218 binding sites for the respective target genes were cloned into pMIR-REPORT expression vector for further luciferase analysis.
Dual luciferase protocol
Manufactured by Promega
Sourced in United Kingdom
The Dual Luciferase Protocol is a laboratory technique used to measure the activity of two different luciferase reporter enzymes within a single sample. This protocol allows for the simultaneous quantification of the activities of two distinct reporter systems, providing a normalized measurement of gene expression or other cellular processes.
Automatically generated - may contain errors
4 protocols using dual luciferase protocol
1
Regulation of 3'UTR targets by miR-218
2
Transient Plasmid Transfection Assay
3
Characterizing Wnt Signaling Activation
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HEK293T cells were transduced with lentivirus coding for the pBARls reporter (Biechele & Moon, 2008 ) and with Renilla Luciferase as a control to generate a Wnt‐βcatenin signaling reporter cell line. To measure F4L5.13 activation, we transduced this cell line with lentivirus coding for human FZD4 protein. For luciferase assay, 2 × 105 cells were seeded in each well of 24‐well plates for 24 h prior to stimulation. The following day, F4L5.13 protein was added, and following 15–20 h of stimulation, cells were lysed and luminescence was measured in accordance with the dualluciferase protocol (Promega) using an Envision plate reader (PerkinElmer). For TSPAN12 dependency, HEK293T‐TopFlash cells were seeded at 0.5 × 106 cells in a 6‐well plate and the next day cells were transfected with FZD4, LRP5 and with either GFP or TSPAN12. Cells were transfected using Lipofectamine 2000 according to the manufacturer’s instructions. The next day, cells were passed in a 96‐well plate with around 35,000 cells/well, and after 5 h, cells were treated with either F4L5.13 or recombinant NDP overnight. After 16 h, cells were lysed, and luminescence was measured.
4
Wnt-βcatenin Signaling Reporter Assay
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HEK293T cells were transduced with lentivirus coding for the pBARls reporter (Biechele and Moon, 2008 ) and with Renilla Luciferase as a control to generate a Wnt-βcatenin signaling reporter cell line. 1–2 × 103 cells in 120 µl were seeded in each well of 96-well plates for 24 hr prior to transfection or stimulation. The following day, FLAg or Ab protein was added, and following 15–20 hr of stimulation, cells were lysed and luminescence was measured in accordance with the dual luciferase protocol (Promega) using an Envision plate reader (PerkinElmer).
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