Fluorescence and absorbance detection of ROS

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Regulation of gene expression and maintenance of genome integrity are fundamental to every organism. Gene activation is needed to express a protein and when that protein is no longer required the gene is turned off. In embryos these events are coordinated and allow naive cells to adopt new fate. Understanding gene regulation is important because often these genes are turned on later in the adult, leading to a number of maladies. DNA is subject to chemical modification and error-prone replication leading to an accumulation of damage and genetic mutations. Numerous agents also influence gene regulation and DNA integrity. For instance, Reactive Oxygen Species (ROS), which are by-products of a cell's ability to metabolize oxygen and produce energy, can damage DNA, proteins and lipids. Cells have mechanisms to prevent ROS damage and quite recently we learned that cells use ROS to initiate signaling cascades that regulate gene expression that benefit the host. Measuring ROS levels under normal conditions, in different genetic backgrounds or at times following environmental stress is thus important and if we can relate these levels to gene activity and genome integrity, much will be learned about how ROS function in cells. Before we can do this, however, we must be able to measure ROS levels and quantify the activity of genes, the level of DNA damage and the frequency of mutations. Towards that end, detection methods were developed to measure ROS levels, DNA damage and gene activity. Although different protocols are required, the instrumentation for detection and quantification is quite similar. At the heart of the methodology is our GloMax Multi-Detection System that was funded by NSERC two years ago. This luminometer has the capability to accurately measure photons of light, with the amounts detected proportional to the amount of gene activity. The request to purchase the fluorescence module to measure the level of ROS and having a dedicated UV-absorbance module to quantify protein expression and peroxide levels will certainly add to our dedicated state-of-the-art equipment, providing our two labs and several trainees with a modern tool to address relevant questions pertaining to a broad spectrum of biology.