Carl Peters, Ph.D., BMG LABTECH, Cary, NC, 07/2013
The PHERAstar FS has been certified as ready to perform the DLR assay from Promega
Instantaneous detection of Firefly and Renilla luciferase is possible due to simultaneous measurement and two onboard reagent injectors
To study the regulation of gene transcription, the DLR or Dual-Luciferase Reporter Assay is commonly used. The two luciferase enzymes used in DLR, Firefly and Renilla are employed in a two-step reaction (Figure 1). The Firefly and Renilla luciferase signals are measured consecutively with intervening quenching of the Firefly luciferase.
Fig. 1: Bioluminescent reactions of Firefly and Renilla
Three criteria must be fulfilled in order to attain DLR certification.
The first criterion indicates whether the second injection step, which contains the Stop and Glo® reagent, has completely quenched the Firefly luciferase reaction. This is called the quenching experiment. To pass the second criterion or consistency experiment a relative standard deviation of less than 5% (%CV) must be maintained by the instrument at two different concentrations of Firefly and Renilla. Finally, the third criterion is the tubing adsorption experiment which indicates the effect the tubing used in the instruments injectors will have on the DLR assay over time. This effect should be minimal.
In order to attain DLReadyTM certification, experiments were performed using the PHERAstar FS (Figure 2).
Fig. 2: The PHERAstar FS- BMG LABTECH’s high performance, multimode reader for HTS
Two injection steps are employed by the dual luciferase assay to carry out the fast reactions. In the first injection the Firefly substrate is delivered in the Luciferase Assay Reagent II (LAR II). The second injection consists of the Stop and Glo® buffer which delivers a Firefly reaction quencher and the Renilla substrate. To quantitate Firefly luminescence followed by Renilla luminescence two sequential 12 second reactions are performed (Figure 3).
Fig. 3: Dual Luciferase Reactions In the first step, to initiate the Firefly luciferase reaction, Luciferase Assay Reagent II (LAR II) is injected. The injection of Stop and Glo® in the second step starts the Renilla Luciferase reaction while quenching the Firefly.
The PHERAstar FS was set up with the following parameters:
Read Mode: Well Mode
Optic Module: LUM plus
Positioning Delay: 0.2 sec
Measurement start time: 0.0 sec
No. of intervals: 48
Interval time: 0.50 sec
Injection speed: 220 µl/sec
Injection start times: 0 and 12 sec
Focal Height: 15.0 mm
Optic used: Top
Aperture spoon: none
Promega documentation, instructions and guidelines were followed for the performance of each of these experiments. While each of the criteria conform to these guidelines there is slight variation from running the kit in its entirety.
Summing of the relative luminescence units over two ranges was performed for data calculation:
Range 1: 2.5 – 11.5 secs or cycles 6 – 24 (Firefly luminescence)
Range 2: 14.5 – 23.5 secs or cycles 30 – 48 (Renilla luminescence)
Criterion 1: Quenching of >10,000 Firefly/Renilla
The quenching seen on the PHERAstar FS for recombinant Firefly luciferase (0.3 ng/mL) was greater than the 10,000 fold required for DLReadyTM status. In order to calculate this; Firefly luminescence was divided by Renilla luminescence (no Renilla was used).
Fig. 4: Criterion 1- Graph showing Firefly luciferase quenching taken from MARS evaluation software (>10,000 fold [n=24])
Criterion 2: Consistency showing < 5% CV
For part 1 of criterion 2, 15 X Firefly to Renilla luciferase concentration was used, while part 2 used 30 X Renilla to Firefly. The CV for Firefly luminescence was 1.52% and Renilla luminescence exhibited a 1.76% CV in part 1. Furthermore, Firefly luminescence exhibited a 1.58% CV and the CV for Renilla was 0.56% in part 2 (Figure 5).
Fig. 5: Criterion 2- Part 2 of consistency is shown graphically with data from MARS data evaluation software (CV’s for Firefly and Renilla = 1.58% and 0.56% respectively [n=24])
Criterion 3: Tubing Adsorption show < 5% CV after 10 minutes
Similar to criterion 2 part one; 15 X Firefly to Renilla was used for this test. Twelve replicates were run followed by twelve more replicates with an intervening 10 minute wait to test for possible tubing adsorption. As with the other tests the % CVs are less than 2 and therefore clearly within the criterion (Table 1).
Table 1: Criterion 3 – Tubing Adsorption shows little change after 10 minutes
Based on the results published in this application note, the PHERAstar FS microplate reader has been granted DLReadyTM certification. The PHERAstar FS is a multifunctional reader suitable for HTS applications. Other instruments from BMG LABTECH which have previously attained DLReadyTM certification by Promega include; the Galaxy, Optima and Omega series of readers. The NOVOstar plate reader with micropipettor and the CLARIOstar, which features an Advanced Monochromator are also certified DLReadyTM.