Dual-Light™ Luciferase & β-Galactosidase Reporter Gene Assay System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 8 × 10^4 cells/well	Dilute Galacton-Plus substrate 1:100 in Buffer B. Add 25 μL of Buffer A to each well. Within 10 min, inject 100 μL of Buffer B (containing Galacton-Plus substrate). After a 1-2 sec delay, read the luciferase signal for 0.1-1 sec/well Incubate for 30-60 min at room temperature.

Upstream tips
Seed 8 × 10^4 cells/well
Protocol tips
Dilute Galacton-Plus substrate 1:100 in Buffer B. Add 25 μL of Buffer A to each well. Within 10 min, inject 100 μL of Buffer B (containing Galacton-Plus substrate). After a 1-2 sec delay, read the luciferase signal for 0.1-1 sec/well Incubate for 30-60 min at room temperature.

Publication protocol

The Hep3B and HEK293 cells were seeded at 8 × 104 cells/well in 12-well plates containing 1 ml of complete medium having 10% FBS. All the transfections were optimized and performed using siPORT NeoFx transfection reagent (Ambion) as per the manufacturer's recommendations. In brief, 1.5 μl of NeoFX transfection reagent was mixed with 48.5 μl OptiMEM (Invitrogen, 11058021), and the mixture was incubated at room temperature for 10 min. miR-31, miR-584 mimics, or its inhibitors (Ambion) (50 nM) were diluted in 50 μl of OptiMEM and then added into the NeoFX transfection agent. The final mixture was incubated at room temperature for another 10 min and used for transfection. A scrambled mirVana miRNA mimic negative control, purchased from Ambion (cat. #4464058), does not have any binding site for eukaryotic 3′-UTRs and was used as a negative control. Mutated miR mimics were also purchased from Ambion. Sequences of mutated miR mimics are shown in Fig. 1. Mock transfections were performed in the absence of additional miRNA mimics. The cotransfection experiments were performed with 100 ng of pMIR/Luc reporter constructs. The pMIR-REPORT β-galactosidase reporter control vector was used to normalize the transfection efficiency. After 48 h of incubation, the cells were washed with 1× ice-cold PBS, and the luciferase and β-galactosidase activities were determined by Dual-Light Luciferase and β-galactosidase Reporter Gene Assay System (Life Technologies) as per the manufacturer recommendations.

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