Myoneurin Troubleshooting and RNA Isolation

26 April 2023

By: Aveya O’Donnell, Madisyn Jarvey, Gustav Pieper

Our group has spent the semester researching the brain and gene expression of Green Anole Lizards (Anolis carolinensis) during the breeding and non-breeding seasons. The gene that our group has chosen to focus our research on is Myoneurin (MYNN). Myoneurin is a regulatory protein gene that is a transcription factor, which regulates the function of other genes.

Aveya and Madisyn capturing an image of their gel electrophoresis on the Bio-Rad gel doc

This process has been quite a journey for our group. We spent most of the semester troubleshooting with three primer sets, which were unsuccessful. After putting our primer’s PCRs through gel electrophoresis multiple times, we kept getting faint bands and primer dimers. We ran temperature gradients to see if our optical annealing temperature was just naturally higher, but that did not seem to play a role in the results we were getting. We then tried a concentration gradient in hopes that we would see darker bands in the right band size area, but that gave us solely primer dimers as it caused the primers to just bind to themselves.

Since our results with those first three primer sets were not successful, two weeks ago our group had to order three new primer sets. It was not ideal since at that point in time we only had about four weeks of the semester left, but fortunately, they arrived quickly. We were then able to create PCRs and start troubleshooting for those primer sets four, five and six. Our results turned out better. After running our first gel, we observed that primer sets four and five had the most potential. The bands were strong and there were no primer dimers. We then conducted two more PCRs on primer sets four and five and the gel for those turned out good as well. We moved on to PCR cleanup and sent our samples off for DNA sequencing.

Gus inserting Lizard brain tissue samples into the refrigerated centrifuge

In the meantime, while all the troubleshooting was going on with our primers, we were introduced to RNA isolations. We practiced the isolations first with three liver tissue samples so that we could better understand how the process worked before moving on to actual lizard brain tissue. The first time we performed on a liver sample, it was hectic. In the past, we had not done anything as fast-paced. Thanks to the practice we had with the liver sample, the brain samples went smoothly. When we ran gels for our RNA isolations, we had two bright and distinct bands, which alludes to the fact that it was successful. We took 1 microliter of our samples and put them into a Nano-Drop to get our concentration and purity. For concentration, we are looking for numbers close to 100. For purity, we want to see numbers close to 2, and our results gave us numbers close to both of these.

Overall, this semester has taught us a lot. It helped us realize that we are not going to get perfect results on the first try and one of the main components of science is trial and error. It taught us patience and teamwork along with the scientific aspect of simple research. We got introduced to new lab techniques and new forms of research. Moving on, we plan to review our DNA sequencing results and then next semester perform a cDNA synthesis and qPCR to quantify gene expression between the breeding seasons.

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