Renilla Firefly System
Renilla Firefly System - Related Videos
04:07
A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells
0 Views •
2025
This video demonstrates an assay to study the translation regulation in poxvirus-infected cells. Uninfected and virus-infected cells were co-transfected with mRNAs encoding for firefly luciferase (Fluc) and Renilla luciferase (Rluc), with the Fluc mRNA containing a 5' Poly(A) leader sequence. Upon adding bioluminescent substrates on lysed cells, measure the signal from both the luciferases to identify the translational advantage of the Fluc mRNA in virus-infected cells, conferred by the leader...
11:34
High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses
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Cited by 14 •
2014
In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.
11:02
Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
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Cited by 1 •
2016
With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.
08:48
Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants
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Cited by 15 •
2016
Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.
12:55
High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
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Cited by 45 •
2014
We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.
FAQs
Normalization is important because raw luminescence can change for reasons unrelated to the regulatory effect being tested. Dividing firefly activity by Renilla activity provides a relative measurement that reduces the influence of differences in transfection efficiency, cell number, and sample handling. The resulting comparison is more reliable than interpreting firefly signal alone.
Signal separation depends on substrate specificity. Renilla luciferase oxidizes coelenterazine, whereas firefly luciferase uses D-luciferin together with ATP, magnesium, and oxygen. Measuring the reactions sequentially lets both activities be quantified from one sample while retaining distinct chemical sources of light. This design supports within-sample comparison rather than requiring separate samples.
A change in raw firefly luminescence does not by itself identify whether gene regulation or experimental variation caused the difference. If the Renilla reference also varies, transfection efficiency, cell number, or sample handling may contribute to the result. Comparing firefly activity with Renilla activity helps account for these influences before interpreting regulatory effects.
Researchers measure both reporter signals from the same sample using sequential substrate reactions, record the luminescence produced by each reporter, and calculate firefly activity relative to Renilla activity. Keeping the measurements paired is central to the internal-control design because the resulting ratio links the experimental reporter to variation present in that sample.
In immunology and infection studies, the system can examine promoter activity, host-pathogen signaling, transcription-factor responses, and regulatory effects caused by cytokines or microbial components. These applications connect reporter output with changes in gene-expression control, allowing investigators to compare how immune-related or infection-related signals influence regulatory activity under the conditions being tested.
A normalized reporter value provides a comparative measure of gene-expression activity that incorporates an internal reference. It can help distinguish differences associated with the regulatory process from variation in transfection efficiency, cell number, or sample handling. In infection and immune signaling experiments, this improves interpretation of responses linked to promoters, transcription factors, cytokines, or microbial components.