Title

Validation of Ninein as an Ethanol-related Quantitative Trait Gene: Reassessment, Design, and Functional Validation of Reference Genes for qPCR Analysis of Brain Tissue in Mice

Defense Date

2020

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmacology & Toxicology

First Advisor

Dr. Jennifer Wolstenholme

Abstract

The increasing use of quantitative real-time polymerase chain reaction (qPCR) as a method for quantifying gene expression has led to an increased demand for standardization of data analysis methods to ensure accurate reporting and robust, reproducible results. The exponential nature of qPCR amplification results in the potential magnification of what are usually very small sources of error. Relative gene expression calculations circumvent this issue by normalizing target gene expression data to within-sample expression of a previously validated, stably expressed reference gene or genes. Multiple studies discussed herein have found that qPCR data are more reliable and reproducible when multiple reference genes are used, and that they are validated prior to use in experiments with new conditions. In this thesis, existing reference genes are evaluated to ensure they meet these criteria in experimental paradigms used frequently in our laboratory. Existing work on ethanol’s anxiolytic-like effects in our laboratory utilized microarrays to identify Ninein as a cis-regulated, quantitative trait gene for these effects in nucleus accumbens (NAc) of BXD recombinant inbred mice and their progenitors, C57BL6/J (B6) and DBA/2J (D2) mice. Contrasting behavioral responses to ethanol in these mouse strains make them a frequent subject of study for determining genetic components underlying those behaviors. In the first data chapter, the case is made for eliminating one reference gene typically used for qPCR data normalization in qPCR experiments assessing strain differences in NAc gene expression in the laboratory, Ppp2r2a. The reference genes subsequently validated for use in qPCR analysis in ethanol-naïve NAc and amygdala of saline and ethanol-treated B6 and D2 mice are then used in an in-depth characterization of Ninein expression in B6 and D2 NAc and amygdala. Furthermore, evidence is provided for the first in vivo observation of murine Ninein transcript variant 6 in adult neural tissue. The data presented make the case for a more thorough re-evaluation of reference genes for future qPCR experiments in the laboratory, as well as a potential mechanism for Ninein’s involvement in variation of anxiolytic-like responses to ethanol in B6 and D2 mice.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

4-25-2020

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