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The Ultimate Guide to FLuc mRNA
FLuc mRNA encodes for firefly luciferase, an enzyme that emits bioluminescence when its substrate, D-luciferin, is added. It is commonly used for gene expression and cell viability assays.
293T cells were transfected with Dluc-wt or Dluc-mut plasmids and treated with pladienolide B (PB, 1000 nM) for 24 h. Dual-luciferase assays were performed to measure Rluc and Fluc luminescence intensity.
Understanding the Function of FLuc mRNA
FLuc mRNA encodes for firefly luciferase, an enzyme that catalyzes the oxidation of luciferin (O2 and ATP dependent), producing chemiluminescence. Originally isolated from the firefly Photinus pyralis, luciferase is commonly used for functional genomics studies due to its high expression level, biodistribution, and stability.
The enzymatic activity of Fluc can be significantly increased by complementation with another gene. This strategy can be used to generate a custom reporter system that can be utilized for a variety of functional studies, including gene regulation, cell viability, and translation efficiency.
To demonstrate the complementation efficiency of this approach, COS-1, N2a, and 293T cells were transiently transfected with pCMV-NFluc-Id or pCMV-MyoD-CFluc vectors alone or cotransfected with both vectors (pCMV-NFluc-Id plus pCMV-MyoD-CFluc). After a 48-hour incubation, enzymatic activity was measured using a luciferin substrate. COS-1 and N2a cells produced significant enzymatic activity, while the CFluc cells had significantly less enzymatic activity.
Northern blot analysis of poly(A)+ RNA isolated from the capped and polyadenylated FLuc-6xT and mut6 mRNAs and their promoterless variants reveals that only the transcripts with the most 3′ end sequence show significant transcription activity. This result is consistent with a model in which miRNA-induced mRNA deadenylation leads to the generation of shorter transcripts.
Understanding the Function of Rluc mRNA
A bioluminescent reporter gene is a DNA molecule that codes for a protein that produces luminescence in response to a substrate. An everyday use for a reporter gene is to monitor gene expression, as the resulting luminescence can be used to assess the transcription level. Several different reporter genes are available, each with advantages and disadvantages.
FLuc mRNA expresses a firefly luciferase (Fluc) protein that responds to the substrate, luciferin, by emitting light. This protein is often used in mammalian cell culture to measure gene expression and cell viability. FLuc is a valuable tool because it does not require a promoter and can be expressed at high levels in cells.
The luciferase protein catalyzes the oxidation of the substrate, generating chemiluminescence that can be detected by a photomultiplier tube (PMT). The luminescent signal luciferin produces is proportional to the cell’s RNA amount. This method is commonly used to study transcriptional regulation in miRNA experiments.
The ability to image Rluc and Fluc expression was demonstrated using a dual-luciferase system. The system uses a mixture of coelenterazine and D-luciferin to visualize the expression of the two genes in living tissues. The kinetics of Rluc and Fluc expression were distinct, and the efficiencies of their reactions differed. Normalization of the results to the chromosomal internal reference genes B2M and ActB reduced the standard deviation but did not eliminate the differences in kinetics.
Understanding the Function of Dluc mRNA
The luciferase gene (luc+) is commonly used in cell biology as a bioluminescent reporter gene because of its ability to detect changes in protein expression. When luciferase is expressed in cells, it will convert substrates like fatty aldehydes and intracellular ATP to chemiluminescence, which can then be measured using luminometers or microscopes. Using a particular luciferase expression system, researchers can monitor the effect of a specific drug on cellular protein production and determine whether it is effective.
Northern blot analyses using a radio-labeled ssRNA probe complementary to the FLuc gene showed that transcription from the cryptic promoter remained active in yeast expressing the luc+ gene under the tightly regulated control of the GAL1 promoter. Moreover, real-time qRT-PCR analysis showed that the production of transcripts complementary to the FLuc CDS increased gradually from its 5′-end to its 3′-end.
Understanding the Function of Pluc mRNA
The firefly luciferase gene from Photinus pyralis is a widely used reporter gene in bioluminescence-based assays. The protein encoded by the flu gene is an oxygenase that oxidizes the heterocyclic substrate d-luciferin to oxyluciferin, emitting luminescence in the wavelength range of 400-620 nm. Luciferase-based assays’ sensitivity, versatility, and simplicity have made the gene popular for thousands of experiments.
One of the most common applications of FLuc mRNA is for sensitive detection of microRNA activity. This is achieved by inserting miRNA-binding sites into the 3′-untranslated region of the bicistronic mRNA or directly within the FLuc coding sequence. In addition, systems that allow for the sensitive measurement of microRNA activity using a single miRNA or siRNA have also been developed.
In mRNA transfection, FLuc mRNA is a proper positive control to assess the efficacy of the cellular transfection process and identify potential problems associated with the experimental conditions.
Moreover, the Fluc mRNA has been stabilized with an RNase inhibitor and delivered as a lipid-modified siRNA. This enables the use of this mRNA in cell culture and animal models without a high risk of off-target effects, a key issue in miRNA research.