As of now, we were able to synthesize RNA for both the wild-type and the I354T mutant. However, we obtained a low yield, meaning we obtained a low concentration of RNA. We are now working on how we can optimize the yield in order to obtain a higher concentration of RNA. RNA is very important because it will be injected into the frog oocytes in order to form a calcium channel protein. Once the calcium channel has formed, then we can perform electrophysiological recordings to compare the currents that go through wild-type calcium channels and calcium channels that contain the mutation.
RNA synthesis is a very delicate and long process. It can easily be contaminated by RNases, which is an enzyme that degrades RNA, that are found in the environment and on human skin. We start the process of synthesizing RNA by first linearizing the wild-type and mutant DNA. Once it is linearized, we purify the DNA by using various chemicals such as phenol/chloroform, chloroform, sodium acetate, and ethanol. This is done to make sure that we have pure DNA and that it is not contaminated. Next, we set up the synthesis reaction using reagents such as the T7 polymerase. After the synthesis reaction, we purify the RNA using similar chemicals that we used for the DNA purification, the only difference being that it is at a different pH. Then, we run our samples on an RNA gel to check the yield and to make sure that it has not been contaminated with RNases. We can tell if it has been contaminated because the bands will appear to be smeared or degraded into separate, smaller bands.
We have gone though this process many times until we finally obtained RNA. RNA is very difficult to make because the process is extremely sensitive since it can be so easily contaminated. Now we are thinking of ways to adjust the protocol so that we can obtain a higher yield of RNA. We think that one reason we may have gotten a low yield was due to the fact that during the synthesis reaction the temperature rose from 37°C to almost 40°C. This sudden rise in temperature may have affected the T7 polymerase, since enzymes are very sensitive to temperature, thus causing us to obtain a low yield. In order to prevent this from happening again, we have to make sure that the incubator we use for the synthesis reaction is set to a steady, constant temperature. Another reason why we may have gotten low yield is because we need to add more T7 polymerase to the synthesis reaction.