Continued qPCR troubleshooting for Beta-Actin and Myoneurin

3 November 2023

By: Madisyn Jarvey, Aveya O’Donnell, and Gus Pieper

Aveya and Madisyn pipetting the SYBR master mix into a cDNA tube

Within the past two weeks, we have continued to troubleshoot our Myoneurin (MYNN)primer set four and beta-actin for our qPCRs and standard curves. So far, our MYNN results have not changed from past runs.Our results continue to show a straight line rather than a curve and a high efficiency. On our most recent standard curve, the efficiency was 8.3982 when it should have been around 2.00. The high efficiency suggests that there are primer dimers presenting themselves in MYNN. However, for beta-actin, we were able to get a good standard curve, a slope, and an efficiency of 1.9291. These results were encouraging, as they showed us that the issues with MYNN are not necessarily due to us performing the qPCR incorrectly. Rather, we may just need to find what works for the MYNN qPCR. Sometimes, a primer set will not work with the SYBR green master mix that is being used for the qPCRs, since it is slightly different than the master mix used for a regular PCR. It also could be a possibility that over time our primers have become degraded or contaminated.

Since we were unsure if MYNN works anymore, we performed a standard PCR on primer set four to confirm that they still bind properly. After analyzing our gel results for the PCR run, we concluded that our primer set does in fact still work. This information will help us determine our next steps in troubleshooting which may include a temperature gradient, adjust primer concentration gradient, or looking into the other two primer sets we made last semester.

Gus filling out his 96 well plate layout sheet

Most recently, because we were successful with our beta-actin standard curve, we moved on to a full qPCR run. For the full run, we used all the cDNA samples that every group in our stream created and ended up with 29 total cDNA samples. We got through the protocol smoothly; however, we ran into an issue with the qPCR machine, and had to discard our plate and the full run. Without any results, we are uncertain how well the process truly went. Our next step is to do another full run with beta-actin so that we can evaluate our results.

Once we get good full run results from MYNN and beta-actin, we can analyze our data using the Excel sheet provided by Dr. Cohen to answer the question we have been working towards all semester: is MYNN expressed differently between seasons in male and female green anole lizards. As we continue to work on the full runs, we will also start working on our end-of-the-year posters that will be presented by our group and the rest of the RISEbio scholars.

We gained a lot from RISEbio in terms of laboratory experience, especially when it comes to techniques like PCR, gel electrophoresis, RNA isolations, and qPCR. Over the last year, we have become better speakers and presenters while also learning to communicate and work better as a group. Overall, the skills we have gained will help us finish the last semester of RISEbio and stay with us as we enter into the professional world.

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