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The post-terminal differentiation fate of RNAs revealed by next-generation sequencing


Advances in RNA sequencing technology allow for potential applications in areas of personal genomics and molecular diagnostics. However, a rate-limiting step of widespread analysis of the transcriptome is the restricted access to viable tissue samples. The hair follicle is an actively regenerating mini organ and the hair shaft is the output of this organ. Here we report on the discovery of hair shafts as a rich, stable source of mRNA, microRNA, and other RNA species that are amendable to extraction and analysis by standard laboratory and next-generation sequencing techniques. Chapter 1 discusses historical methods to assay correlations between genetic differences and phenotypic differences. Chapter 2 argues for the suitability of the hair follicle as a model organ of study. Genetic changes and other health states are known to affect the physical appearance and other properties of the hair shaft. Chapter 3 describes the demonstration of lineage specific RNA in hair shafts and our creation of a small RNA library of short sequences expressed in the human hair shaft. The library serves as a starting point to identify and predict expressed genes that may be biologically relevant. Some of the well represented RNAs are the Let-7 family of microRNAs, keratins, and keratin binding proteins. Other genes known to be specific to the cortex, medulla and cuticle of the hair are also present. Our library contains an abundance of microRNA compared with libraries made from other tissues. Chapter 4 discusses the process of cornification and compares the coverage of hair shaft RNA to that of a viable keratinocyte cell line. This also led us to investigate whether smaller RNAs were more stable than longer RNAs in the hair shaft in Chapter 5. As hair on the human scalp has an unusually long growth phase, we analyzed RNA from hair that grew out over several months. We report that hair segments representing up to a year of growth contain extractable RNA that is amendable to downstream applications. In addition, we find that another keratinzed tissue, nail, also retains RNA. Chapter 6 discusses these findings and potential applications in the field of personalized medicine and retrospective screening

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