For this summer research project, we are in the process of making RNA, which is very delicate and can easily contaminated by RNases. RNA can be degraded by RNases, which are found in the human skin and the environment. Thus, it is critical to change gloves often, clean all of the materials (pipettes, epitubes, etc) with RNase detergent before use and make sure all solutions are RNase free. The process of making RNA starts by linearizing 8μg of the mutagenic DNA overnight at 37°C. The amount we linearize depends on the size of the construct and the concentration of the DNA. If we use a higher concentration of 0.500 μg/μl, then we will use about 16 μl of DNA, which is significantly less than the amount required when you have a lower concentration of DNA such as 0.160 μg/μl. The second step is the DNA purification using chemicals such as phenol/chloroform (pH 8), chloroform, sodium acetate and pure ethanol. This step involves cleaning, neutralizing and drying the samples. After the samples are dried, we start the RNA synthesis reaction using T7 polymerases and other reagents that will optimize the reaction. We found that our hPQ samples are optimized by leaving the synthesis reaction to occur overnight. The next step is RNA isolation and purification using the same chemicals that we used for the DNA purification, however, the phenol/chloroform for this procedure is at a pH of 5.2. This process also involves a drying step, in which the drying time varies from 30 min to 2 hours and depends on the amount of Ethanol that must be evaporated. After the samples are completely dried, we resuspend the RNA in DEPC water and run a RNA gel to check the RNA yield and whether there was RNase contamination, which can be seen as bands smudge and degrade into smaller bands. Following all the procedures as described above, the process of making RNA takes about 3 days before you get the results.
We have tried to make RNA four times this summer. After each trial we analyze and research for any new troubleshooting procedure that would help us optimize the RNA synthesis process. We found that for our human PQ type calcium channel mutants Q1397X and R477H, we can optimize the purification process by adding pre-chilled ethanol, incubating at room temperature for 10 min and incubating at -20 degrees for 20 min. It is quite challenging to make RNA since it is easily degraded by RNase in the environment and any contamination will ruin the process of synthesizing RNA. Due to this challenge in making the RNA, we were not able to start TEVC experiments, in which we inject the RNA into frog oocytes to express the mutations then we inject a current to check the functionality of the Voltage-gated Calcium channels expressed. We were going to test the expression of both mutagenic and Wild Type human PQ channels and how this may reflect in the development of epilepsy.
I have learned to be meticulous and often plan ahead in order to finish all the procedures on time. Since we have to be very careful making the RNA, I have learned to prepare all the necessary materials and equipment on the lab bench and wiping them down with RNase detergent before any of the procedure takes place. This summer research opportunity taught me how to multi-task between various projects. We worked on making other mutagenic constructs by doing transformations, minipreps, testcuts and sequencing. We were able to get four different constructs with the correct mutagenic DNA. I also worked with a smaller beta subunit of the calcium channel and successfully made the mutagenic RNA for the beta-3 E53K by using some of the optimization techniques that we applied to the much larger PQ channel. Thus, there is still hope that we will be able to make RNA for the human PQ type channel by the end of this summer.