Dual-Luciferase® Reporter Assay System From Promega

The Dual-Luciferase® Reporter (DLR™) Assay System is widely used for performing two reporter assays. Each system includes 10 ml Luciferase Assay Buffer II, 1 vial of Luciferase Assay Substrate, 10 ml of Stop & Glo Buffer, 200 ul of Stop and Glo Substrate (50X) and 30 ml of Passive Lysis Buffer (5X). A single sample can be used for the activities of both firefly (Photinus pyralis) and Renilla (Renilla reniformis or sea pansy) luciferases. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) which generates a luminescent signal for a short period of time. Then addition of Stop & Glo® Reagent to the same sample causes quenching of the previous reaction and initiation of the Renilla luciferase reaction. Both assays can be completed in about 30 secs/sample using a luminometer. The DLR™ Assay provides rapid quantification of both reporters, either in transfected cells or in cell-free transcription/translation reactions.

I have been using this kit for the last 3 years. Overall, I am quite happy using this kit. I generally do transfections in 24-well plates using non-small cell carcinoma cell lines. (A427 and A549). I generally introduce 1 ug of DNA luciferase construct along with 50 ng of renilla luciferase construct into each well using Qiagen’s Effectene™ Transfection reagent. After removing the growth media, I rinse the cultured cells in 1X PBS. At this point, the plate can be stored at –80ºC. When we are ready to proceed, we put the plate at room temperature for adding passive lysis buffer. For lysis, 100 ul of 1X passive lysis buffer (5X passive lysis buffer is provided with the kit) is added to each well and the 24-well plate is then kept in the shaker for about 15 minutes at room temperature. Then 20 ul of each sample lysate is used for the assay. Here I add 50 ul (though the recommended volume is 100 ul) of Assay Reagent II and Stop & Glo® Solution for measurement in the luminometer. So briefly, add 20ul lysate in the luminometer tube followed by addition of 50 ul LARII. Measure firefly luciferase activity. Then dispense 50 ul of Stop and Glo Reagent followed by the measurement of Renilla luciferase activity.

Overall, I am very happy with this kit. The results are consistent when I use equal numbers of cells to begin with. When the same number of cells are not used for transfection, I have noticed that the results follow the cell number; absolute values vary from experiment to experiment.