RapidReporter^®

While reporter gene assays, most commonly luciferase, have become an important tool for studying signal transduction pathways and gene expression, the assay itself is limited by the fact that basal activity of the cloned promoter results in accumulations of both the luciferase mRNA and protein. The slow clearance rate of these pre-existing reporter molecules substantially delays and dilutes the measurable response to stimulation or repression. In the case of repression, this is because the reporter mRNA continues to produce new (and long-lived) reporter protein long after transcription has stopped. In the case of stimulation, this is because the short, rapid increase in luciferase that occurs in response to whatever stimulation is being tested has proportionally little impact on the high steady-state levels already present. As a result, with standard reporter gene assays, transient or relatively minor effects are hidden (Figure 1) and kinetic assays are inaccurate.

Figure 1: RapidReporter “unmasks” hidden effects.

HeLa cells were transiently transfected with pRR-High-NFκB and pGL3 vector containing NFκB and plated in 96-well plates. Twenty-four hours post-transfection, cells were stimulated with 10 ng/ml TNF-α. At the indicated time points, the cells were measured for Gaussia luciferase (pRR) and firefly luciferase (pGL3). Because of the increased sensitivity imparted by its double destabilizing elements, NFκB's natural oscillation from the cytosol to the nucleus during activation is observed by RapidReporter.

Double destabilization improves response

To solve the problem of long half-life proteins, some assays incorporate a protein-destabilizing element in the vector, causing the reporter protein to degrade more rapidly. Destabilizing the protein, however, only partly addresses the problem as protein clearance rates are also dependent on the half-life of the reporter mRNA. As long as the pre-existing reporter mRNA remains intact, it continues to produce new reporter protein. To address this issue, the patented RapidReporter method utilizes vectors that include both protein AND mRNA destabilizing elements. This makes the assay much more responsive to both increases and decreases in transcriptional activity. Thus, RapidReporter provides a more accurate measurement of the actual transcriptional activity of the cloned promoter (Figure 2).

Figure 2: More accurate kinetics of induction by isoproterenol.

293 cells were transiently transfected with pRR-High-CRE, pRR-Low-CRE and a pGL3 vector containing CRE (CREB Response Element) and plated onto 96-well plates. Twenty-four hours post-transfection, cells were stimulated with 4 µM isoproterenol. At the indicated time points, the cells were measured for Gaussia luciferase (pRR-High-CRE & pRR-Low-CRE) and firefly luciferase activities (pGL3-CRE).

RapidReporter^® advantages

• Increased magnitude of response provides greater separation between active and inactive compounds, greatly reducing false positives
• Faster response to stimulation enables the detection of transient effects and drugs that decompose rapidly
• Ability to detect weak to moderate stimulations that are not found by other assays, due to their high basal background levels
• Increased productivity as the fast response enables you to run more assays, and to identify more actual leads

Your choice of stringency

RapidReporter vectors are offered in two different stringencies. The pRR-High vector provides the lowest background possible because it contains more and stronger destabilization elements and thereby provides maximum responsiveness. The fold induction of stimulated vs. non-stimulated samples is highest using pRR-High. pRR-High vectors are ideal in cases where your stimulation will produce a weak effect. In contrast, the pRR-Low vector contains fewer and weaker destabilization elements. While this lowers the level of the fold-induction observed, it enables weaker basal signals to be detected. pRR-Low vectors are ideal for use when a low signal strength is expected, such as with cells that transfect at a low efficiency or when testing a promoter with weak activity in the cells of interest.

Pre-made vectors & complete assay kits

In addition to empty RapidReporter vectors, Active Motif offers a variety of pre-made vectors that already contain widely studied promoters (see Ordering tab). All vectors are available separately or in complete assay kits, which include a positive control vector, Lysis & Assay Buffers and a substrate optimized for Gaussia luciferase.

Please see the Contents tab for details on what is included in each RapidReporter product configuration.

Contents & Storage

RapidReporter Luciferase Assays (Cat. Nos. 33001 & 33002) provide 100 or 1000 reactions of 5X Lysis & 1X Assay Buffers and Gaussia Substrate; these kits do not contain any RapidReporter vectors.

RapidReporter Luciferase Assays with vector (Cat. Nos. 33004, 33006, 33008, 33010, 33012, 33014, 33016 & 33018) contain 10 µg of the appropriate vector, 100 reactions of Lysis & Assay Buffers and Gaussia Substrate, and 10 µg of a positive control vector driven by the EF1α promoter.

RapidReporter Luciferase Vectors (Cat. Nos. 33003, 33005, 33007, 33009, 33011, 33013, 33015 & 33017) provide only 10 µg of either empty or pre-made RapidReporter vector; these kits do not contain any buffers, substrate or positive control vector.

Store all RapidReporter reagents at -20°C; please see manual for details. All reagents are guaranteed stable for 6 months when stored properly.

* RapidReporter is covered under U.S. Patent No. 7,157,272 and various other patents worldwide, is a registered trademark of, and is sold under license granted by GeneStream Pty Ltd. Purchasers are subject to a Limited-use Label License, which can be downloaded at the Documents tab. Commercial entities and all users who wish to perform high-throughput screening will be required to enter into an End User License Agreement with GeneStream following a 6-month evaluation period. Please contact your Active Motif sales representative or one of our Technical Service departments for details.