Developing Taqman Open Array Assays to test circadian clock gene expression
Standing
Undergraduate
Type of Proposal
Oral Research Presentation
Challenges Theme
Open Challenge
Faculty Sponsor
Phillip Karpowicz
Proposal
Circadian rhythms are internal mechanisms present in nearly all animals that time behaviour and physiology to occur in 24-hour oscillation periods. We attempted to develop TaqMan Open Array assays to detect conserved regions of clock genes. We initially targeted 13 circadian clock genes and 3 control genes. Primers were designed for regions of homology in Salmonids and Chinook salmon (Oncorhynchus tshawytscha), and following their design were tested using Chinook cDNA by SYBR Green qPCR. It was possible to design primers targeting Salmonid cDNA for all genes; these were used to construct TaqMan Open Array chips successfully. To test whether these were able to detect circadian gene expression, we investigated Chinook muscle tissues of fish in four environmental conditions. We examined Chinook salmon parr entrained in 4 different photoperiods: 12-hour Light:12-hour Darkness (12:12 LD), 8:16 LD, 16:8 LD, and 24 DD (complete darkness). The final condition is a test of circadian rhythmicity which will persist in the absence of photoperiod. As expected, circadian clock genes oscillated in a 24-hour manner in the 12:12 LD condition, but these are altered in 8:16 LD, and 16:8 LD, in a gene-specific manner. Importantly, these genes persisted in DD conditions, demonstrating that the circadian clock is functional, and gene expression observed is not due to stochastic changes. Our ongoing work includes deeper analysis on the changes in clock genes’ expression between these different photoperiod conditions tested, and how these influence muscle tissue growth and morphology. Using these parameters, overall Chinook health will also be assessed.
Grand Challenges
Viable, Healthy and Safe Communities
Developing Taqman Open Array Assays to test circadian clock gene expression
Circadian rhythms are internal mechanisms present in nearly all animals that time behaviour and physiology to occur in 24-hour oscillation periods. We attempted to develop TaqMan Open Array assays to detect conserved regions of clock genes. We initially targeted 13 circadian clock genes and 3 control genes. Primers were designed for regions of homology in Salmonids and Chinook salmon (Oncorhynchus tshawytscha), and following their design were tested using Chinook cDNA by SYBR Green qPCR. It was possible to design primers targeting Salmonid cDNA for all genes; these were used to construct TaqMan Open Array chips successfully. To test whether these were able to detect circadian gene expression, we investigated Chinook muscle tissues of fish in four environmental conditions. We examined Chinook salmon parr entrained in 4 different photoperiods: 12-hour Light:12-hour Darkness (12:12 LD), 8:16 LD, 16:8 LD, and 24 DD (complete darkness). The final condition is a test of circadian rhythmicity which will persist in the absence of photoperiod. As expected, circadian clock genes oscillated in a 24-hour manner in the 12:12 LD condition, but these are altered in 8:16 LD, and 16:8 LD, in a gene-specific manner. Importantly, these genes persisted in DD conditions, demonstrating that the circadian clock is functional, and gene expression observed is not due to stochastic changes. Our ongoing work includes deeper analysis on the changes in clock genes’ expression between these different photoperiod conditions tested, and how these influence muscle tissue growth and morphology. Using these parameters, overall Chinook health will also be assessed.