I was originally approached about this project at the end of the summer, around August, and have been working on it since. The goal is to do a quantitative analysis of relative mRNA expression for four known escape genes across the FCG mice model. The four chosen genes are known as X-linked genes, which are those that consistently escape X chromosome inactivation (XCI) across tissues and in various species, including humans and mice. These genes have higher expression in XX cells compared with XY cells, and are important contributors to sex differences in health and diseases This mouse model allows us to generate XX and XY mice, each with either ovaries or testes: XXF (XX + ovaries), XYF (XY + ovaries), XXM (XX + testes), and XYM (XY + testes).

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The project began with pre-harvested samples of FCG mice cochlea which were stored at -80°C from the right inner ear. Total RNA was isolated from the tissue samples using the RNeasy® Lipid Tissue Mini Kit and QIAzol® Lysis Reagent. The quantity and purity of RNA (ng/µL) was determined through absorbance data using the NanoDrop™ Spectrophotometer​​. If purity was below 1.7 then RNA precipitation would then be used to increase the purity. Primers were designed using Using the ApE (a plasmid editor) by M. Wayne Davis, for use in qRT-PCR. The criteria for primer use are flanking end lengths of 19-28 nucleotide bases long, a guanine/cytosine percentage between 40-60%, but as close to 40% as possible, and a melting temperature between 57-60°C​. Using iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, 1725120) , the primers were checked for validity for use in qRT-PCR through CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). The total RNA that was previously synthesized is then converted back into cDNA using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, 18080051). Quantitative reverse transcription polymerase chain reactions (qRT-PCR) were performed for all the genotypes and primers​