Promega
Dual-Luciferase® Reporter 1000 Assay System
E1980
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Primary cilia are tiny microtubule-based structures that project from the cell surface. These structures serve as the signaling hubs for the Sonic hedgehog signaling. The goal of this experiment is to determine if 3T3 cells (mouse origin) respond to sonic hedgehog ligand treatment in a Gli transcription factor (the ultimate factors in the sonic hedgehog pathway that induce target gene induction) reporter assay.
Luciferase reporter assay
3T3 cells
3T3 cells were transfected with a 8xGli binding site driven Firefly luciferase construct and a Renilla luciferase construct. Confluent cells were serum starved to induce primary cilia and then treated with a sonic hedgehog conditioned medium for 24 hours. Cells were washed in 1x PBS and lysed in 1x passive lysis buffer (supplied in the kit). 5 µl of lysate was pipetted into a 96 well white plate and luciferase activity was read on a Synergy H1 Multi-Mode reader (Biotek) with a dual injection mode. 20 µl of LAS reagent was added and read for 10 sec followed by 20 µl of stop & Glo reagent and read for 10 sec.
3T3 cells have poor transfection efficiency and so the values might be low. However, one should definitely see a nice increase in Firefly luciferase activity upon ligand addition
3T3 cells nicely respond to sonic hedgehog ligand and the fold change can be anywhere between 8-20.
None
Very easy to use
Expensive
I never had a trouble with this kit and it is a very straight forward assay kit
Product Name: Dual-Luciferase Reporter 1000 Assay SystemSupplier Page