Dual Luciferase Reporter (DLR) assay certification

Introduction

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 FLUOstar^®, and LUMIstar^® Omega as well as on the CLARIOstar and PHERAstar FS which achieved DLReady™ certification.

Assay Principle

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

Instrument settings

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 %.

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.