Next Generation Sequencing Study of Circadian Changes in Transcriptome of Rhesus Macaque Pineal Gland and Retina

Purpose: We performed an NGS study on the circadian changes in Rhesus Macaque retina and pineal gland transcriptome in order to elucidate novel elements in the primate circadian clock. Methods: Total RNA from retina and pineaal glands of Rhesus Macauqes euthanized at 4 timepoints (Dawn, Day, Dusk and Night) was deep sequenced, in triplicate, using Illumina HiSeq2000. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2. Results: We discover a variety of novel genes that show circadian activivty in both retina and pineal gland of the Rhesus Macaque. A particular set of genes involved in the production of N-acetyltryptamine in Rhesus Macaque retina appears to be responsible for the circadian oscillations of the concentrations of N-acetyltriptamine in the plasma, thereby accentuating N-acetyltriptamine as a retina derived signaling molecule in primates. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Retinal and Pineal gland Total RNA reads were generated by deep sequencing, in triplicate, using Illumina HiSeq2000. Control samples consisting of same amount of RNA extracted from corpus callosum, frontal cortex, cerebellum, thalamus, hippocampus, caudate, medulla, pituitary, heart, liver, lung, kidney, skeletal muscle, small intestine, testis and ovary, at mid-day (12 P.M.) and midnight (12 A.M.).