Loading my One Millionth qPCR Plate

Author: Karma Hine | Major: Biology | Semester: Spring 2023

My name is Karma Hine and I graduated summa cum laude from the Fulbright College of Arts and Sciences in Spring 2023, where I majored in Biology and double minored in medical humanities and psychology. While fulfilling the requirements for my honors thesis, I discovered an unexpected passion for biological research that led to my decision to pursue an MD-PhD after graduation. To earn more experience in a research environment and come to final conclusions for my undergraduate research, I decided to earn an M.S. in Cellular and Molecular Biology at the University of Arkansas before enrolling in medical school. Since my junior year of college, I have worked as an undergraduate researcher in Dr. Christian Tipsmark’s physiology lab, which is part of the Biological Sciences department, where I was welcomed into the world of fish physiology and hormonal regulation.

I initially contacted Dr. Tipsmark during my freshman year after scrolling through a list of faculty members with undergraduate research labs. He specialized in fish biology with a focus in osmoregulation, and I was initially drawn to the lab because of my love for fish! After sending him an email, he gave me a tour of the laboratory and introduced me to some electric fish as well as his tank full of Atlantic killifish, the model organism he was using for experiments.

Allie McFarland, the master’s student in his laboratory, was focused on hormonal effects on the expression of cellular effectors in the gill and opercular membrane of these fish. The osmoregulatory machinery behind the composition of membrane and tight junction proteins in the intestine is largely unknown, and because Allie was doing similar experiments with the gill, Dr. Tipsmark decided that I would conduct similar experiments on intestinal segments! I was very happy with this decision because the intestine is much easier to remove, cut, and work with than gill arches, which are very small and fragile. The larger organ segments also had more RNA, which meant I would be more likely to successfully extract good-quality RNA as I was training in the laboratory.

I worked alongside Allie, shadowing and then repeating her gill experiments with my intestinal segments. The science behind osmoregulation in these euryhaline fish, which can adjust to large fluctuations in salinity, is relevant to medical science, as many intestinal diseases stem from an inability to properly regulate ion movement. The cellular machinery behind osmoregulation in these fish is similar to that of our own regulation, which makes them a good model organism for studying phenotypic response. For example, the hormone prolactin stimulates ion retention and water secretion in both humans and euryhaline fish.

During the spring grant term, I met with Dr. Tipsmark weekly and came up with a comprehensive plan to finish collecting, analyzing, and documenting data for my honors thesis. I was in the laboratory with a graduate supervisor for several hours a week extracting RNA, synthesizing complementary DNA (cDNA), loading qPCR plates, and analyzing the results using prism and excel. The qPCR machine uses a very expensive fluorescent chemical called SYBR™ Green Master Mix coupled to a primer set to look for the DNA sequences of specific osmoregulatory proteins. Using this method, I could observe the relative mRNA expression of these proteins in response to the concentration of cortisol and prolactin the intestinal segments were treated with.

The biggest learning curve I faced while conducting research was learning how to properly pipette! When you’re working with volumes as low as 1μL, a very small inconsistency can have big effects on standard error and mess up the significance of your data. My favorite part of the research was sitting down and analyzing data to look for significant effects. Ultimately, I discovered that the Na⁺/K⁺/2Cl^– cotransporter (nkcc2), which is an ion regulator also found in the human intestine, was upregulated in response to treatment with cortisol.