Related Experiment Video
Updated: Jun 1, 2026
Using Human Differentially Expressed Gene Lists to Perform Downstream Pathway Enrichment Analysis and Target Prioritization
Published on: October 3, 2025
An exercise to estimate differential gene expression in human cells
1Department of Medical Laboratory and Radiation Sciences, Burlington, Vermont 05405; DNA Microarray Facility, University of Vermont, Burlington, Vermont 05405. mchaudhr@uvm.edu.
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.
Insights
Researchers developed an accessible reverse transcription-polymerase chain reaction (RT-PCR) method to quantify gene expression changes. This technique effectively measures ionizing radiation-induced alterations in human cells for educational and research applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Gene expression varies across tissue types and is linked to organ function.
- Altered gene expression patterns are observed in disease, in response to treatments, infections, and environmental exposures.
- Understanding molecular-level disease mechanisms requires studying differential gene expression.
Purpose of the Study:
- To develop a simplified, non-radioactive method for examining and quantifying differential gene expression.
- To create an educational exercise for college students using reverse transcription-polymerase chain reaction (RT-PCR).
- To investigate ionizing radiation-induced changes in human cell gene expression.
Main Methods:
- Developed a differential gene expression assay based on RT-PCR.
- Isolated total RNA from human cells at various time points post-ionizing radiation exposure.
- Converted RNA to cDNA and performed multiplex PCR using gene-specific primers and beta-actin standards.
- Quantified gene expression differences using free imaging software.
Main Results:
- Successfully established an easy-to-perform, non-radioactive RT-PCR technique for differential gene expression analysis.
- Demonstrated the method's ability to detect ionizing radiation-induced gene expression changes in human cells.
- Validated the use of beta-actin as an internal standard for accurate quantification.
Conclusions:
- The developed RT-PCR method is suitable for educational purposes, enabling students to perform gene expression analysis.
- This technique offers a versatile and accessible approach for investigating differential gene expression across various organisms and experimental conditions.
- Provides valuable insights into molecular responses to environmental factors like ionizing radiation.
