- Promega´s DLR assay had been validated on the Omega, CLARIOstar® and PHERAstar®FS microplate readers from BMG LABTECH
Table of contents
The Dual-Luciferase® Reporter Assay or DLR is widely used to study gene transcription and regulation. The DLR assay is a two step reaction that uses two luciferase enzymes, Fireﬂy and Renilla (Figure 1). The Fireﬂy reaction is initiated, followed by its quenching and the subsequent initiation of the Renilla reaction.
The dual measurement of these two enzymes allows for an experimental measurement and a transfection control measurement to be done at the same time. This dual reporting of each sample allows a quantitative result based on the normalization of the Renilla luciferase (transfection control). More information on the DLR assay and its certiﬁcation requirements are available on Promega’s website at www.promega.com and in the technical manual.
The certiﬁcation process consists of 3 parts: Quenching, consistency and tubing adsorption.
The ﬁrst criterion is the quenching experiment. This will indicate whether the Stop and Glo® reagent, added in injection step 2, has successfully quenched the Fireﬂy luciferase reaction initiated by Luciferase Assay Reagent II which was added in injection step 1. The second criterion is the consistency experiment. These experiments determine whether a relative standard deviation of less than 5% (%CV) can be maintained by the instrument at two different concentrations of Fireﬂy and Renilla luciferase. The tubing adsorption experiment is the third criterion. This experiment shows whether over time the tubing used in the instruments injectors will have an effect on the DLR assay.
These 3 experiments were conducted on the POLARstar®, FLUOstar® and LUMIstar® Omega as well as on the CLARIOstar and PHERAstar FS which achieved DLReady™ certiﬁcation.
The dual luciferase assay is a fast reaction with 2 injection steps, one for the Fireﬂy substrate (Luciferase Assay Reagent II or LAR II) and one for the Stop and Glo® buffer which contains the Fireﬂy quencher and the Renilla substrate (Figure 2).
The reaction requires an injection and a measurement for 12 seconds (to quantitate the Fireﬂy luminescence) and then another injection and another 12 second measurement (to quantitate the Renilla luciferase).
Materials & Methods
- White, ﬂat-bottom 96-well Costar® plates
- Promega’s DLR certiﬁcation kit
- Recombinant Fireﬂy and Renilla luciferase provided by Promega
|Omega series||CLARIOstar||PHERAstar FS|
|Method||Well Mode Kinetic, Top optic|
|Optic settings||Emission: lens||Emission: |
(520 – 620 nm)
|Positioning delay||0.2 seconds|
|Number of intervals||48 Inverval|
|Inverval time||0.5 seconds|
|Injection start time||0 and 12 seconds|
|Injection speed||220 or 230 µl/second|
These experiments were performed as described in the Promega Instrumentation Certiﬁcation documentation. Each test varies slightly from running the kit as a whole. Each of the 3 criteria for certiﬁcation were run according to Promega’s guidelines.
For data calculation, the relative luminescence units are summed over two ranges:
- Range 1 - Fireﬂy luminescence (3.0-12 seconds).
- Range 2 - Renilla luminescence (14.5-23.5 seconds).
Results & Discussion
Criterion 1: Quenching of >10,000 Fireﬂy/Renilla
Recombinant ﬁreﬂy luciferase exhibited quenching that was >10,000 fold (DLR requirements) (Figure 3). This was calculated by dividing blank corrected Fireﬂy luminescence by blank corrected Renilla luminescence (no Renilla was used in this experiment).
Criterion 2: Consistency showing < 5% CV
For criterion 2, a 15 X Fireﬂy to Renilla luciferase concentration was used for part 1, while a concentration of 30 X Renilla to Fireﬂy was used in part 2. The %CV values were below 5 % for all tested BMG LABTECH instrumentation.
Table 1: Criterion 2 – Consistency has to be < 5 %.
|%CV, n = 24||Consistency Part1||Consistency Part2|
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 3 and therefore clearly within the criterion (Table 2).
Table 2: Criterion 3 – Tubing Adsorption shows little change after 10 minutes, for n=12.
|RLU (after 10 min)||1.030E7||1.9||3.793E6||2.5|
|RLU (after 10 min)||6.91E6||0.5||1.762E5||2.0|
|RLU (after 10 min)||1.536E7||1.1||1.536E7||1.8|
The Omega series of microplate readers as well as the CLARIOstar and the PHERAstar FS from BMG LABTECH has been granted DLReady™ certiﬁcation based on the results published in this application note.