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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
Published on: June 16, 2022
Isotopic assays for reporter gene activity
R E Kingston1, J Sheen, D Moore
1Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Current Protocols in Pharmacology
|October 4, 2011
Summary
This unit details two radioactive reporter gene assays: chloramphenicol acetyltransferase (CAT) and human growth hormone (hGH) radioimmunoassay. These methods quantify gene expression and protein secretion in transfected cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Reporter gene assays are essential tools for studying gene expression.
- Radioactive detection assays offer high sensitivity for quantifying biological molecules.
Purpose of the Study:
- To describe two established radioactive reporter systems: chloramphenicol acetyltransferase (CAT) and human growth hormone (hGH).
- To provide a detailed overview of the methodologies for these assays.
Main Methods:
- Chloramphenicol acetyltransferase (CAT) assay: Measures gene expression by quantifying the enzymatic activity of CAT in cell lysates using radioactive substrates.
- Human growth hormone (hGH) radioimmunoassay: Quantifies secreted hGH using a two-site radioimmunoassay with radiolabeled antibodies and immune complex capture.
Main Results:
- The CAT assay allows for the assessment of gene transfection and expression levels.
- The hGH radioimmunoassay accurately determines the amount of secreted hGH in cell culture medium.
Conclusions:
- Both CAT and hGH reporter systems provide reliable methods for analyzing gene expression and protein secretion.
- Radioactive detection assays remain valuable tools in molecular and cell biology research.
Related Concept Videos
02:11Reporter Genes
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...
Commonly used reporter...
03:31Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...