In our search for multiplexed assays that increase both assay speed and sensitivity we have developed
a panel of three ultrasensitive luciferase reporter-Cypridina luciferase (emission max 463 nm), an improved green-emtiting Renilla luciferase (emission max 527 nm) and a Red=emitting Luciola Italica firefly luciferase (emission max 617 nm). The red-emitting firefly luciferase is a 1000 times brighter than it's native counterpart). The green-emitting Renilla luciferase mutant is about 35-40 times brighter than native Renilla luciferase (human codon optimised best commercial version). These luciferases thus offer increased sensitivity in screening applications involving analysis of weak promoters or hard-to-transfect cells. The green variant of Renilla luciferase also offers improved stability of the luminescent signal both in vitro and in vivo. The triple luciferase assay reagents and protocol described here permit analysis of all three luciferases in the same sample of cell lysate and thus increases speed of the assay. The assay system provides flexibility of assaying the three luciferase reporters in three formats as follows:
i) All three luciferases can be assayed in the same sample of cell lysate by sequential addition of Cypridina luciferase, Firefly luciferase and Renilla luciferase assay reagents to the same sample of cell lysate. In the format the Cypridina luciferase reagent is added first and the sample assayed for Cypridina luciferase activity. Twenty minutes later the Cypridina luciferase signal decays completely and the firefly luciferase assay reagent is added to measure the firefly luciferase acitivty. This is followed by the addition of the Renilla luciferase assay reagent (with an inhibitor of firefly luciferase) which quenches the firefly luciferase and enables measurement of Renilla luciferase activity. Thsi is the most preferred format of the assay as it improves speed fo the assay and improves sensitivty
- The three luciferases are assayed using a single solution and spectrally resolving the luciferaes activities using appropriate filters.
3)The three luciferases can be assayed in separate samples of supernatant or cell lysate using the assay reagents for each luciferase.
This triple luciferase assay systems is particularly useful in studying multiple pathways within the same cell and profiling responsiveness at different times (without cell lysis) . For example, using NFkB
and CREB response elements to follow apoptosis and GPCR profiling in the same group of transfected cells.