title: An exercise to estimate differential gene expression in human cells.
abstract: The expression of genes in cells of various tissue types varies considerably and is correlated with the function of a particular organ. The pattern of gene expression changes in diseased tissues, in response to therapy or infection and exposure to environmental mutagens, chemicals, ultraviolet light, and ionizing radiation. To better understand the disease at the molecular level, the alterations in gene expression can provide clues to identify the cellular pathways involved. There are a wide variety of techniques that have been developed to study differential gene expression. Most of these methodologies are technically challenging. We developed an exercise to examine and quantify differential gene expression based on reverse transcription-PCR. Our goal was to have this technique successfully performed by college level students. This technique is easy to perform and does not require the use of radioactive substances. We examined the ionizing radiation-induced changes in gene expression in human cells. After exposure to ionizing radiation, total RNA was isolated at 4, 8, 12, and 24 h. The RNA was converted to cDNA and subjected to PCR amplification using gene-specific primers and commercially available β-actin internal standards in a multiplex format. The differences in gene expression were quantified with freely available imaging software. This technique should have a wide application to investigate differential gene expression in a variety of organisms and under various experimental and treatment conditions.
authors.affiliation: Department of Medical Laboratory and Radiation Sciences, Burlington, Vermont 05405; DNA Microarray Facility, University of Vermont, Burlington, Vermont 05405. mchaudhr@uvm.edu.
authors.forename: M Ahmad
authors.initials: MA
authors.lastname: Chaudhry