1 December 2023
By: Anna Wilcox, Emma Rieper, and Ellie Malone
These past two weeks we have been conducting our final experiments on our three genes, ZYG11A, HS6ST1, and DIO2. All the while putting our finishing touches on our final poster for end of the year presentations of our research. DIO2, a gene that we recently added, is a gene that turns deactivated T4 thyroid hormone into activated T3 thyroid hormone. These hormones both play a role in the production of metabolism.
One of the major procedures that we have completed is a standard curve. A standard curve analyzes our NTC (no template control), 5-standard, 1-standard, 0.2-standard, and 0.04-standard tubes. The NTC does not contain cDNA, while the standards contain a specific diluted amount. All of these tubes contain a specific amount of the master mix created. This standard curve run tells us if our standards, as stated, were pipetted properly and accurately. Also, if the cDNA that we used was working and not denatured. This line of samples and our NTC is used as a standard to allow us to compare our 24 cDNA samples and their expression levels.
After we received good results for at least one standard curve, we started to run full run qPCRS, or quantitative PCRs. To complete a full run qPCR, we first made Int-1, Int-0.2, and Int-0.04. These all contained water and a diluted amount of cDNA from the previous tube. Next, we created our NTC and standard tubes. We then created a master mix, which contains all of the necessary components to amplify these 24 cDNA samples with the genes forward and reverse primers. Included in the master mix is a liquid called PowerUp SYBR Green 2x Mix. This is the base of the master mix which allows for a lot less area for pipetting error. The rest of the master mix includes both forward and reverse primers and water. This mixture was pipetted into 24 tubes and then the correlating cDNA was added to each of these tubes. These were then pipetted into a 96-well plate and ran through the qPCR machine. Both ZYG11A and HS6ST1 have each had two standard curves and one successful full run qPCR conducted on them and have shown good results. DIO2 had one standard curve and one successful full run qPCR which showed promising results.
After analyzing our results, we concluded that none of our genes had a significant change during the breeding vs non-breeding season in male or female green anole lizards. We created a poster and presented these findings, along with our journey to them, at a poster session this past week. We will be presenting this information again in April at the URS conference on campus.