What is a dual reporter assay?

The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc).

What is a reporter assay?

A bioluminescent reporter assay consists of both a luciferase reporter enzyme and a detection reagent that provides the enzyme substrate. When the reporter enzyme and detection reagent are combined, the light emitted is proportional to reporter gene expression levels and is detected using a luminometer.

What is the purpose of a reporter assay?

The main purpose of the reporter gene assay is to investigate the promoter of a gene of interest, i.e. the regulation of its expression. This can be done by linking the promoter of interest to an easily detectable gene, such as the gene for firefly luciferase, which catalyzes a reaction that produces light.

Is luciferase a reporter?

The ARE/LUCPorter(TM) reporter cell line is a stably transfected MCF7 cell line which expresses an optimized Renilla luciferase reporter gene (RenSP) under the transcriptional control of the antioxidant response element (ARE).

What is Rlu in luciferase assay?

Marshall University. Basically, you can consider RLU as light intensity, which is a number assigned by the instrument—luminometer to your sample. It has NO UNIT. LAR II measure luciferase activity in your sample, which represents the activity of promoter etc.

What is reporter gene example?

For example, yeast reporter genes include CUP1, a gene that enables yeast to grow on copper-containing media, URA3, a gene that kills yeast when growing on 5-fluorouracil, and ADE1 and ADE2, two genes that synthesize adenine.

What is a reporter strain?

Reporter strains have proven to be powerful tools to study Mycobacterium tuberculosis (Mtb) physiology. Transcriptional and translational reporter strains are engineered by fusing a readout gene, encoding a fluorescent, luminescent or enzymatic protein, downstream of a promoter or in-frame with a gene of interest.

What is a luciferase reporter assay?

A luciferase reporter assay is a test that investigates whether a protein can activate or repress the expression of a target gene using luciferase as a reporter protein (Carter & Shieh, 2015). This bioluminescence directly corresponds with the effect of the protein on expression of the target gene.

What makes a good reporter gene?

The ideal reporter gene should be absent from the cells used in the study or easily distinguishable from the native form of the gene, assayed conveniently, and have a broad linear detection range.

How does a luciferase reporter work?

A Luciferase Reporter Assay. When this protein activates transcription, the cell will produce luciferase enzyme. After the addition of a lysis buffer and a substrate, a luminometer quantifies the luciferase activity. If your protein activates the expression of the target gene, the amount of signal produced increases.

How do reporter genes work?

Reporter genes are genes that enable the detection or measurement of gene expression. They can be fused to regulatory sequences or genes of interest to report expression location or levels.

Which is the best dual reporter assay to use?

Previously, we described some of the advantages of using dual-reporter assays (such as the Dual-Luciferase®, Dual-Glo® Luciferase and the Nano-Glo® Dual-Luciferase® Systems). Another post describes how to choose the best dual-reporter assay for your experiments. For an overview of luciferase-based reporter gene assays, see this short video:

What are dual spectral luciferase reporter assays used for?

With these types of experiments, called dual-spectral luciferase reporter assays, two luciferase enzymes are simultaneously expressed and detected in a single sample. Dual-spectral luciferase reporter assays are ideal for:

How are reporter assays used in molecular biology?

REPORTER ASSAYS Reporter Genes Genetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. They are widely used in pharmaceutical and biomedical research and also in molecular biology and biochemistry.

How is the firefly luciferase reporter assay measured?

The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a luminescent signal lasting at least one minute. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is initiated simultaneously by adding Stop & Glo® Reagent to the same sample.