Analysis of Gene Expression Following Oxidative Stress

The modulation of gene expression by cellular stress is a universal phenomena that has been described in organisms ranging from bacteria to animals to plants, and in response to a wide range of stress agents including heat shock, glucose deprivation, oxidants, radiation, and heavy metals (1 ). For oxidative stress alone, many genes have been identified whose steady-state mRNA product levels are modulated by a multitude of oxidant stress agents (1 ). These genes were identified using various techniques that are still popular, such as two-dimensional gel electrophoresis, subtractive hybridization, differential display (DD), restriction fragment differential display (RFDD), serial analysis of gene expression (SAGE), and gene arrays; as well as other techniques that have lost their popularity, including one-dimensional protein gel electrophoresis and differential hybridization (1 ). Differential display (DD) is a polymerase chain reaction (PCR)-based technique that requires only small amounts of sample, is relatively sensitive, and able to identify both induced and reduced species (2 –3 ). Its main drawbacks are a high number of false-positives and products that are strongly biased for the ’3’ untranslated region of the mRNA. These drawbacks have been addessed by a more recent technique called restriction fragment differential display (RFDD), which uses restriction enzyme-generated cDNA fragments as PCR templates (1 ). This modification allows for the use of much higher temperature and more specific annealing conditions as well as the ability to generate products that include the coding region and ’5’ untranslated region.