Using Real-Time RT-PCR to Measure the Changes in the Expression of Caenorhabditis Elegans Genes Involved in the Regulation of "Free" Iron and Defense Against Oxidative Stress.

Steven Pauff

Date of Award

2008

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

Reactive Oxygen Species, Antioxidant Gene Expression, Reverse Transcription-Polymerase Chain Reaction

Area of Concentration

Chemistry

Abstract

The Oxidative Theory of Aging suggests that some of the detrimental effects of aging are a result of the degradation of cellular components through oxidative attack by reactive oxygen species (ROS) produced during ATP synthesis within the mitochondrial electron transport chain. Recent work at California State University, Fullerton has focused on the use of Fe(III) electron paramagnetic resonance (EPR) spectroscopy to quantify the change in mobilized ("free") iron amounts within the model organism Caenorhabditis elegans undergoing treatments associated with ROS production. C. elegans employs a variety of gene products to regulate iron concentrations and resistance to ROS-produced oxidative damage; real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) can be used to detect changes in the expression of these genes. In this work, C. elegans wild type (N2) and mutant (mev-1) strains underwent four treatments known to either result in ROS production or resist ROS production. These conditions were 4 hour heat shock (ROS producing), 12 hour high and low temperature exposure (ROS producing), treatment with 100 mM paraquat (ROS producing) and ionic manganese supplementation (ROS resisting). mRNA from these worms was collected and utilized in real-time RT-PCR relative quantification experiments to determine whether the expression of these genes was correlated with the increase in "free" iron observed in EPR measurements. RT-PCR results from the heat shock and temperature exposure experiments did not correlate with the changes in "free" iron observed in the Fe(III) EPR results. Interestingly, the PCR results from these tests seem to indicate that temperature is involved in the regulation of C. elegans gene expression. mev-1 worms undergoing paraquat treatment showed gene regulation consistent with an increase in ROS production which suggests that Fe(III) EPR could be used as a tool in ROS measurements. Results from ionic manganese treatments suggest that ionic manganese is beneficial to wild type worms by increasing their resistance to oxidative stress; furthermore, resistance appears to increase with increasing concentrations of ionic manganese.

Rights

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

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