Isolating DNA from BANG samples after RNA extraction was complete
Author: Melih Karabacak | Major: Biology | Semester: Spring 2024
My name is Melih Karabacak and I am in the Fulbright College of Arts and Sciences. I am currently a Biology major with a Chemistry minor, and I am conducting research under my mentor Dr. Jeannine Durdik in the Biology department. My future plans are to continue this research until my graduation in Spring 2025, complete my medical school applications over the summer, and then attend a school based on my options after one gap year. This blog is written for my Spring 2024 semester of funding.
During my Sophomore year, I was interested in many different fields under Biology. However, one that stuck out to me the most was genetics, especially in terms of gene editing. As a result, I was motivated to begin my research career and emailed the head of the Biology department for any current faculty that were looking for help in their lab. Through some discussions, I had eventually chosen to take part in Dr. Jeannine Durdik’s research lab. Specifically, I was interested in their topic of choice: understanding the complex relationships between a macrophage’s immune response and its age, temperature, and species.
For the first year I was in Dr. Durdik’s lab, I spent my time educating myself on her lab’s research topics. Primarily, this was achieved through spending time in the lab itself, performing literature review, joining weekly lab meetings, and using my skills in R code to help with statistical analysis. Eventually, I was ready to pick a topic with Dr. Durdik that I could perform my independent research on. After some meetings with her, I eventually decided to do my research on the underlying genetic mechanisms during points of intense metabolic change for stimulated macrophages under normal and fever temperatures.
Through my experiences, I have learned that once macrophages are stimulated, they undergo a specific pattern in metabolic changes for the next six hours; this change is measured in PER (glycolysis) and OCR (metabolic respiration). Furthermore, this pattern can be denoted by four specific points: Basal, Activation, Nitric Oxide production, and Glycolysis peak, together known as BANG. I have also found out that macrophages that were put under fever temperatures had BANG points far earlier and at elevated values compared to those at normal temperatures. This correlates with the tendency for macrophages to have improved function at fever temperatures, however, the underlying genetic mechanism for this process was still unknown.
Thus, I wanted the aim of my experiment to be able to gather RNA from macrophages at each of the BANG points (at both normal and fever temperatures), and conduct genetic analysis for the functional molecules and metabolic pathways that were being utilized to improve immune function. Furthermore, in recent years, it has become more difficult for people to reach ideal fever temperatures, primarily due to decreasing average body temperatures in the past 100 years and this temperature naturally decreasing with increasing age. However, artificial fever temperatures (ie. a warm blanket or an incubator) have shown to still improve immune responses. As a result, by understanding the genetic pathways that contribute to the relationship between fever temperatures and improved macrophage function, I will also be able to get better insight on how to improve the stimulation of this relationship and assist hospital patients.
During last semester, I was able to harness the skills I needed to complete my experiments, and understand the importance of planning out and refining procedures to efficiently complete runs. Furthermore, with the help of Dr. Durdik and another graduate student, Kenneth Taylor, we had done our first rounds of experimentation, but ultimately had to repeat it since we did not get sufficient data.
This past semester, I have been working diligently to complete the experimentation portion of my research project, and begin moving into analysis and discussion. However, this proved to be a bit more challenging than anticipated. After our initial BANG run, another one was completed close to the beginning of the semester. However, this also proved to be a failure as the timing of our extractions were not entirely correct. Later on, after more detailed planning and careful following of extraction protocols, we had a successful BANG run and extracted RNA from all four points at two different temperatures. Furthermore, after the RNA was isolated and washed, it was packaged and sent over to a sequencing company.
At the end of this semester, I am now waiting and excited to see what sequencing data we get from our RNA extractions. Additionally, in order to prepare to analyze this data, I have started to complete some literature review again and understand how RNA sequencing data is supposed to look like. Lastly, since we had extracted solutions left from our BANG run, I have started isolating the DNA and proteins from all of four of these points as well, allowing us to complete more experiments and analysis in the future.
Like last Fall, I ran into many challenges this semester as well. However, I was fortunate enough to have my mentor and her graduate student to guide me at every step of my research project. One big challenge I had faced again was communication and coming into my lab consistently. However, I was able to recently fix this by scheduling semi-weekly meetings with my mentor, and having block out times each week for when I absolutely will be able to show up in the lab. Furthermore, an important lesson I learned about working in a research lab like this is that you should not be afraid to ask questions on anything you are uncertain of. As a result, I have been able to keep more up to date with what is happening in the lab, and improve my research skills in general. For the future, I will be continuing this research project by completing analysis of the sequenced RNA data. Moreover, once this is completed, I hope to have my research published and present at state and national conferences.