By: Sarah Oberstar and Kaity Shaffer
24 September 2024
We are back in the lab, ready to continue our research from last spring. In the final weeks of the previous semester, we finalized and sequenced our primers for KCNJ4 and NR1D2. We also finished isolating RNA. This semester we are using our isolated RNA and turning it into cDNA. We are also preparing to take the next step in our research, qPCR. This includes practicing standard curves and serial dilutions.
We started by converting our isolated RNA to cDNA through cDNA synthesis. RNA is very unstable and must be stored at –80 degrees Celsius. By converting RNA to DNA, it becomes much more stable. We don’t have to store it at such low temperatures, and it becomes easier to work with. In total, we synthesized six different samples for the research stream to use. After all six samples were converted into cDNA, we performed a PCR using a primer we know worked and gel electrophoresis to make sure that the band sizes looked correct. This makes sure we have quality cDNA to use for future experiments. We have done this process twice, so far five out of the six samples have turned out well.
The next step in our research will be qPCR. This week in the lab we just practiced the protocol using water and dye. We started by making a serial dilution. We used three tubes to make our concentrations. Each tube was more diluted than the last. This series will be similar to the one we will use to make a standard curve for when we do the real qPCR. Next, we did some dilution calculations just to make sure we understood the math. Lastly, we practiced the qPCR protocol. In the past we have used microcentrifuge tubes to do PCR, this time we got to use a 96-well plate. Each of us got a map and used colored water to fill said plate. This was a very cool practice to do, we are very excited to do the real qPCR.
All in all, we synthesized cDNA from RNA and verified its quality using PCR and gel electrophoresis. Five out of the six samples have so far been successful. We need to keep working on the last sample in the coming weeks. We practiced the qPCR protocol and are looking forward to applying it in future experiments. We learned many new things about qPCR, the protocol, and the process. We learned that we will have to be patient and focused when completing the real protocol. We discovered how serial dilutions and standard curves relate to qPCR.
