Stress-responsive gene expression and protein adaptation: analysis with CFX96 real-time PCR detection system

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Research in my lab explores the biochemical adaptations that allow animals to survive in extreme environments. We analyze amazing natural phenomena including hibernation, freeze tolerance, cold hardiness, estivation and anoxia tolerance. All of these are linked by the use of hypometabolism (transitions to torpid or dormant states) to achieve long term viability under stressful conditions. Our research not only identifies key principles of metabolic regulation that underlie hypometabolism and life extension across the animal kingdom but it has important potential applications in biomedicine because methods for inducible dormancy could greatly improve procedures for storage of human organs removed for transplantation. Research by students in my lab (currently 17) relies heavily on two major experimental approaches - studies of stress-responsive gene expression (changes in mRNA transcript levels, microRNA regulation of mRNA translation, transcription factor activation, etc.) and studies of enzyme adaptation (kinetic properties, structural stability, stress-induced covalent modification, etc.). PCR-based gene expression studies have become critically limited by our inability to do quantitative (Q)-PCR (that is now the analytical standard for publication) that requires a real-time fluorescence detection system. The present application will solve this problem with the addition to our core analytical instrumentation of the CFX96 Real-Time PCR Detection System (Bio-Rad). This instrument will provide state-of-the-art Q-PCR capabilities and, very importantly, will give us a new, highly sensitive method for analyzing enzyme/protein stability: differential scanning fluorimetry (DSF) to detect thermal unfolding of proteins using fluorescent dyes. With this method we can study a range of influences on enzyme stability (effectors, cryoprotectants, covalent modification) comparing stress tolerant vs intolerant species with micro-amounts of proteins, very precise thermal control and the ability to compare multiple conditions in a single 96 well plate. Addition of the CFX96 will truly give us a quantitative leap in analytical capacity that will strongly aid our goals of discovering the molecular mechanisms of how animals work.