This summer, I worked in the Department of Otorhinolaryngology under the guidance and supervision of Dr. Erle Robertson. The Robertson lab primarily studies Kaposi’s Sarcoma herpesvirus (KSHV) and Epstein Barr Virus (EBV), two human oncogenic gammaherpesviruses that are causative infectious agents of various pathologies and other human malignancies. My project focuses on the BHRF1 microRNA (miRNA) cluster encoded by EBV genome expressed in viral positive cell lines. Early studies have found the function of miRNA to be indirect or direct degradation of mRNA. My project aims to identify and confirm potential viral targets of these BHRF1 miRNAs in order to determine if these interactions play a role in maintaining viral latency, thus contributing to tumorigenesis.
During my time in the lab this summer, I gained an incredible amount of insight into the inner workings of conducting my own project. The beginning of the summer saw a revision to my experimental plan for this project, perhaps one of the most crucial aspects of research. This project employs various molecular biology techniques, including western blots, RT-qPCRs, and luciferase assays. In order to initiate some of the early studies of my experimental plan, I had to confirm that I was able to reactivate two EBV-positive cell lines, 293T EBV GFP Bac and B95.8 using 12-O-tetradecanoylphorbol-1 3-acetate (TPA) and butyric acid, two chemical reagents that have been found to induce a number of viruses into the lytic phase. By demonstrating that these viruses were reactivated, I would then be able to quantify each BHRF1 miRNA to better understand the stage of the viral life cycle where each are most upregulated. This will then give me a better sense of the dynamic quantitation of the BHRF1 miRNAs at the different stages of the viral life cycle.
An experiment that I anticipated to take a fraction of the summer led to some difficulties that resulted in me spending most of my summer addressing the problems and ended up consuming most of the summer. Some of the problems I encountered included incorrect concentrations of TPA and butyric acid used to reactivate the viruses, insufficient levels of cell confluency upon reactivation, and primary antibodies which were not well characterized for my western blot. This summer required me to think about the technical demands and the questions that should be asked when conducting biomedical research. The time I spent in Dr. Robertson’s lab this summer has been an invaluable experience that has made me even more eager and excited to see this project through during the academic year.
I am grateful to Dr. Robertson for allowing me to work in his lab and his careful guidance throughout my process of revision and experimental troubleshooting. I would also like to thank the College Alumni Society Research Grant for supporting me this summer.