4 APRIL 2019
By Nicole Michealson, Marc Nganteh & Curtis Hanson
Introduction
Anolis carolinensis, known as green anole lizards, are native to North America, located primarily in the southeastern part of the continent. The RISEBio Brain and Behavior Research Stream focuses on this species because of the behavioral differences in non-breeding versus breeding seasons. The breeding season occurs between the spring and summer, during the months between April and September. The behavioral differences of anole lizards occur in males during their breeding season, where they will defend their territory by bobbing their heads at rivals and displaying their brightly colored dewlap. The male’s dewlap is also used to attract females during the breeding season. Besides behavioral differences, both male and female anole lizard gonads increase in size significantly during the breeding season. For our research, each group chooses a specific gene to see if it is expressed differently during the breeding versus the non-breeding seasons. Our group chose the thyroid stimulating hormone beta (TSHB) gene, which is produced in the base of the brain in a structure called the pituitary gland. This gene produces a hormone (thyroid stimulating hormone) that is critical for the growth and function of the thyroid gland. More importantly, these hormones play a role in growth and brain development, which could be an influence on anole lizard’s reproductive and behavioral differences in the different seasons.
Research
We started off by picking our gene, which was the TSHB gene. We were supposed to design 3 primers with certain requirements found through the National Center for Biotechnology Information’s website. However, only two primer sets met the requirements we needed to work on. When the primers arrived, we reconstituted them to allow us to begin working on Polymerase Chain Reaction (PCR).
Then we went on to perform our Polymerase Chain Reaction (PCR) to amplify a specific fragment of our gene’s DNA, known as the amplicon. We proceeded to do gel electrophoresis and gel imaging to visualize our results from the PCR. To our disappointment, the results for our primer sets showed no clear bands, and the water controls we used showed primer dimers. We were encouraged by our professor and mentors that it is common for PCR and gel electrophoresis to not show strong results the first few times of doing it, due to the lack of experience. The fact that we also did PCR on beta actin gene that showed positive results also gave us hope to carry on, because we knew with certainty beta actin would show bands around 120 base pairs.
In the past two weeks of research, we have continued to do PCR and gel electrophoresis to make sure our primer sets are displaying strong bands in the right product length of 84 and 86 base pairs. We have started to change our variables to see if our results will become clearer under different circumstances. We have begun doing a temperature gradient between 58 °C-68 °C during the primer annealing stage, which is the stage that allows primers to attach to a certain location on a DNA template. There were difficulties during this, the Quick-load TAQ we use for our master mix ran out, so we could only run one of our primer sets. We also ran our gel electrophoresis in the wrong direction, so we were not able to accurately see our results. Luckily, since we only used 10 microliters of our primer set to do the gel, we used the rest to correctly do another gel electrophoresis and imaging. We found that our primer set for TSHB showed the clearest band at the temperature of 61.8 °C. We then ran another PCR with a master mix of the TSHB for that given temperature and the TSH primer set we were not able to do a temperature gradient for the PCR with. The results from the gel electrophoresis and gel imaging showed faint bands for the TSH primer set at all different temperatures, and the TSHB primer set showed an evident band at the temperature 61.8 °C once again. This week we just finished up doing our PCR cleanup for the TSHB primer set, where we cleaned the 9.8 microliters left from our PCR sample using different buffers. We used the nanogram to measure the concentration and purity of the TSHB sample. This process is to ensure our sample is not contaminated before sending it in. The concentration was 6.6 ng/μL and the purity level is 1.71 (1.8 is the ideal purity). We will send these results in to confirm that the PCR is amplifying the right gene.
Future Research
After running our DNA through PCR and confirming that it worked through gel electrophoresis, we need to isolate the RNA from tissue samples. DNA contains an extraordinary amount of genes, however, not all of the genes in DNA are expressed. Most genes remain dormant so that it can later be copied but are usually never used aside from that. mRNA can be thought of as showing what genes are being expressed at the time. This aspect of time is vital to our research because we are researching how our gene affects Anolis carolinensis during the breeding and non-breeding seasons. Performing RNA extraction is difficult because RNA in general is pretty unstable, meaning that much care must be given to be successful in the procedure. After checking the quality of the extracted RNA to see if it’s usable, we can hopefully start seeing how our gene affects Anolis carolinensis throughout its life.
References
Wikipedia Foundation, Inc. (2019, March 11). Carolina Anole. Retrieved from: https://en.wikipedia.org/wiki/Carolina_anole
National Library of Medicine. (2019, March 19). TSHB Gene. Retrieved from: https://ghr.nlm.nih.gov/gene/TSHB
