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Comparison of Assay Genie Dual Luciferase Reporter Assay (MORV0010) vs Promega (E1910): Performance Evaluation of Quenching Efficiency and Auto-luminescence

Validation Data
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Introduction

The Assay Genie Dual Luciferase Reporter Assay Kit is a robust and economical tool designed to measure the activity of both Firefly and Renilla luciferases from a single sample. This kit is ideal for studying gene regulation and signaling pathways in cells transfected with reporter plasmids. Firefly luciferase serves as the primary reporter using luciferin as a substrate, while Renilla luciferase acts as an internal control using coelenterazine as a substrate, helping to normalize variations in transfection efficiency and cell number.


In luciferase-based assays, two critical performance parameters must be optimized: quenching efficiency and auto-luminescence. Quenching efficiency is important for ensuring that the signal from Firefly luciferase is sufficiently quenched before detecting Renilla luciferase, preventing signal overlap and ensuring accurate dual luciferase measurements. On the other hand, auto-luminescence refers to the background luminescence from Renilla luciferase in the absence of substrate activation. 

High auto-luminescence can obscure true signal detection, leading to less accurate results.


Therefore, both factors are crucial for maintaining the sensitivity and specificity of the assay, directly impacting the reliability of the experimental data. By comparing these two parameters between Assay Genie and Competitor P, researchers can better understand the performance of each kit in producing reproducible, high-quality data.

1. Quenching Efficiency

To assess quenching efficiency, we measured the luminescence of Firefly luciferase before and after adding the Stop and Reaction Buffer in three independent experiments. Quenching efficiency ensures that residual Firefly luminescence is minimized before detecting Renilla luciferase, preventing interference between the two signals.

Table 1: Quenching Efficiency - Firefly luciferase luminescence values before and after the addition of Stop & Reaction Buffer.

Assay Genie

Assay Genie

Promega

Promega

Test

Before Stop & Reaction Buffer

After Stop & Reaction Buffer

Before Stop & Reaction Buffer

After Stop & Reaction Buffer

Test 1

1,201,733

1,137

1,018,940

710

Test 2

1,122,958

937

1,010,801

439

Test 3

1,190,229

1,355

1,023,784

519

Average

1,175,306

1,143

1,017,841

556

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(Firefly luciferase before buffer addition) compares initial luminescence values between Assay Genie and Competitor P. Assay Genie showed slightly higher initial values, reflecting its robust signal generation, which is beneficial for detecting gene expression changes.

Graph 1 (Firefly luciferase before buffer addition) compares initial luminescence values between Assay Genie and Competitor P. Assay Genie showed slightly higher initial values, reflecting its robust signal generation, which is beneficial for detecting gene expression changes.

Graph 2 (Firefly luciferase after buffer addition) shows that both kits achieve highly efficient quenching of Firefly luciferase. Competitor P demonstrated slightly lower post-quenching luminescence, with a quenching efficiency of 99.95%, compared to 99.90% for Assay Genie. Nevertheless, both kits minimize residual luminescence sufficiently to allow for accurate Renilla luciferase detection.

Graph 2 (Firefly luciferase after buffer addition) shows that both kits achieve highly efficient quenching of Firefly luciferase. Competitor P demonstrated slightly lower post-quenching luminescence, with a quenching efficiency of 99.95%, compared to 99.90% for Assay Genie. Nevertheless, both kits minimize residual luminescence sufficiently to allow for accurate Renilla luciferase detection.

2. Auto-luminescence of Renilla Luciferase

Auto-luminescence refers to the inherent background luminescence from the Renilla luciferase system, even without substrate activation. Low auto-luminescence is crucial for preserving the assay's sensitivity and ensuring that the detected signal is primarily due to Renilla luciferase activity, rather than background noise.

Graph 3 visualizes these results. Both Assay Genie and Competitor P maintain very low auto-luminescence values, with Assay Genie averaging 680 relative luminescence units (RLU) compared to Competitor P's 639 RLU. This indicates that both kits provide minimal background signal, ensuring sensitive Renilla detection with negligible interference.

Table 2: Auto-luminescence (background) values of Renilla luciferase.

Assay Genie

Promega

Test

Auto-luminescence (Renilla)

Auto-luminescence (Renilla)

Test 1

873

686

Test 2

592

683

Test 3

576

549

Average

680

639

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Conclusion

The comparison between the Assay Genie Dual Luciferase Reporter Assay Kit and Competitor P demonstrates that both kits provide exceptional performance in key areas:


Quenching Efficiency:

Both kits exhibited high quenching efficiency, with more than 99.9% of Firefly luciferase activity quenched, ensuring accurate detection of Renilla luciferase without residual interference from Firefly luminescence.


Auto-luminescence:

Both kits maintain very low auto-luminescence levels, ensuring that background signals do not compromise assay sensitivity.

In conclusion, the Assay Genie Dual Luciferase Reporter Assay Kit offers performance comparable to that of more expensive competitors, making it a cost-effective solution for researchers studying gene regulation and signaling pathways. Its high sensitivity and reliable results make it an excellent choice for laboratories requiring accurate, reproducible luciferase assays.


For further details on the Assay Genie Dual Luciferase Reporter Assay Kit or to request technical support, please visit our website or contact our team (info@assaygenie.com).

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17th Feb 2025 Colm Ryan

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