Document Type

Thesis

Date of Award

10-2025

School/College

College of Science, Engineering, and Technology (COSET)

Degree Name

MS in Biology

Committee Chairperson

Dr. Shodimu-Emmanuel Olufemi

Committee Member 1

Dr. Desirée Jackson

Committee Member 2

Dr. Ayodotun Sodipe

Committee Member 3

Dr. Roderick Holmes

Abstract

Gene expression is measurable at the RNA and protein levels. It is a process by which genes produce functional products that are either non-coding RNAs or proteins. The non-coding RNAs or proteins of gene expression ultimately affect phenotype. Non-coding RNA (or mRNA) expression is measured by Northern blot or real-time quantitative PCR (RT-qPCR), while Western blot measures protein expression. Both require software to differentiate between the amounts of end products. Statistical analysis is employed to validate any differences among the measured values and to infer a correlation between the end products of various samples. There are several neurodegenerative diseases, including Alzheimer's disease (AD). Widespread damage to neurons, loss of connections to other neurons, neuron death, and improper functions slowly destroy memory and thinking skills and reduce the ability to carry out the simplest tasks. Apart from heritable Alzheimer's disease, other forms of Alzheimer's disease are age-related, unknown genetics, environmental, and lifestyle factors. Massive neuronal cell death occurs in the brains of AD patients through necrotic or apoptotic mechanisms. Cell existence and continuity depend on cell survival, proliferation, and metabolism, and are controlled by AKT, an intermediary major player. AKT regulates cell survival, growth, proliferation, angiogenesis, vasorelaxation, and cell metabolism through the IP3/AKT signaling pathway. Although there are several studies on the IP3/AKT signaling pathway, there are still limited studies on the expression of neuronal protective, survival, and proliferating genes and the microRNAs (miRNAs) that are likely potential biomarkers for predicting AD occurrence in brain samples. Therefore, it is essential to study the gene and miRNA expression to determine potential biomarkers for predicting AD. Hence, this study develops a preferential amplification method and uses a semi-quantitative PCR (sqPCR) to determine the RNA expression of AKT2, NONO, CBR3, CDK2, hsa-miR-200c, hsa-Let-7a, and hsa-miR-1227 in the frontal lobe, precentral gyrus, temporal lobe, AD brain, and normal brain RNA samples. This study aims to demonstrate that preferential primers designed for AKT2V1 primer sets coupled with sqPCR amplification will differentiate between the multiplex and non-multiplex expressed RNA. In addition, comparing sqPCR amplified end products of the preferential primer designed primer sets of multiplexes and the non-multiplex will yield a precise amount of expressed AKT2, NONO, CBR3, and CDK2, including hsa-miR-200c, hsa-Let-7a, and hsa-miR-1227, which will differ among AD brain samples. This research proposes to address gene expression complexities in AD brain samples. The specific aim of this study is to demonstrate that preferential primer design for multiplex primer sets, and non-multiplex primer sets coupled with sqPCR and/or qPCR will yield a precise or close amount of expressed RNA of AKT2V1, other genes, and miRNAs in Alzheimer's disease RNA samples. Therefore, this study develops two hypotheses based on the specific aim. The first hypothesis is that a preferential primer design for multiplex primer sets, and the non-multiplex primer sets used for sqPCR amplification will produce dissimilar amounts of amplified AKT2 RNA and identify an AKT2 primer set suitable for sqPCR. The second hypothesis is that the most preferential amplified expressed AKT2 in hypothesis 1 will show far more differences when compared to the expression of other genes (NONO, CBR3, CDK2) and expression of the miRNAs (hsa-miR-200c, hsa-Let-7a, and hsa-miR-1227) will differ in the brain areas: the frontal lobe (which affects intelligence, judgment, and behavior), the precentral gyrus (which affects memory), and the temporal lobe (which affects language), allowing for the detection of genes and/or miRNAs suitable as biomarkers after sqPCR and proper statistical analysis. In conclusion, the results certify differences in multiplex sqPCR and identify a primer set preferentially amplified among the AKT2V1 primer sets. The RNA expression of AKT2V1, NONO, CBR3, CDK2, hsa-miR-200c, hsa-Let-7a, and hsa-miR-1227 differs among the AD brain samples. This study is the first preferential primer-designed method for designing multiplex primer sets for sqPCR used to identify the most preferentially amplified PCR end products among AKT2 primer sets and detect maximal RNA expression in AD brain samples. This study also identified differences in the RNA expression of AKT2V1, NONO, CBR3, and CDK2, including hsa-miR-200c, hsa-Let-7a, and hsa-miR-1227, using the non-multiplex primer sets. Statistical analysis also validates differences in RNA expression among the genes and miRNAs.

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