Studying Gene Expression of PLD4 and Troubleshooting Primers

17 November 2023

By: Georgia Deml and Rebecca Fasching

Since our last blog post in September, we have made significant progress in each of our genes. We also have had great success with the combination of our lab groups. We have found out that we work very well and efficiently together. We had a lot of work that needed to be done, especially early in the semester but we have managed to make good progress in our research especially with the PLD4 gene. Even without progress there is still much to do before the semester is over.

Rebecca performing a PCR cleanup on STAB2 primers with our TA Nick watching.

 When it comes to gene progression specifically in PLD4, we designed 3 new primers and found that our new primer set 2 worked very well. The new primer set worked well enough to be able to send for DNA sequencing. Our results came back looking good from the sequencing. During the past few weeks, we were introduced to qPCR reactions. This was a learning curve for us and every other group because we were moving from pipetting into a few tubes to now pipetting into a 96 well plate. We had a lab session where we practiced pipetting into the plate with different colored water to get a feel of what a full run would be like. Our first few qPCRs were not full runs because we were still learning and needed good results on a standard curve to be able to proceed onto the full run. PLD4 looked good in the standard curve runs so we were able to recently complete a full run with PLD4. The full run results came back looking good and when we compared the numbers to beta actin, it supported the hypothesis we originally had made for PLD4 that the inflammatory response will be higher in males during the mating season.

Georgia pipetting our PCR mixes into an agarose gel.

While the work with PLD4 has been going along great, there have been some struggles with STAB2. Initially our STAB2 primers were all set to be run with a qPCR as the primer set we were using had been sequenced last semester and came back looking good. However, we began seeing problems with our standard curve runs of STAB2. The efficiencies of the runs were no where near the value we needed to continue to a full run and when run on a gel would only show primer dimers. We began troubleshooting to see if we needed to alter the conditions of the qPCR to get STAB2 to work. We began with a temperature gradient on a normal PCR and saw that the reaction still worked better at the standard 60℃ but still had primer dimers. Next, we tried the PCR reaction with different primer concentrations to no avail. During our second attempt with primer concentrations, we also made a normal PCR using one of the other two primer sets we created last semester. The other primer set came out well on the PCR so we did a PCR cleanup and sent it out for sequencing. If the sequencing looks right with the other primer set we will make another attempt at running a standard curve with STAB2.

Despite the setbacks with STAB2 we have managed to do well this semester. We were able to get the data needed to test our hypothesis for PLD4 and found that our research supports our hypothesis. We do not yet have the data necessary to do the same for STAB2 but we are making progress to get to that point. This semester has been an excellent experience especially with the opportunity to be working with someone new.

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