Up until this week I was practicing taking tissues from mosquito dissections and turning them into RNA. This week I learned how to DNase treat the RNA, convert the RNA to cDNA, perform PCR(Polymerase Chain Reaction), and run gels. I'll explain the theory behind these processes and then discuss my gel pictures!
We are interested in extracting RNA because it is involved in gene expression. When we run DNA that was made from RNA in a gel, we can see how well our inserted gene is being expressed. Recall that genetic material comes in two forms, DNA and RNA. If the whole tissue is taken, we are going to get RNA(what we want) and genomic DNA(what we do not want). Genomic DNA cannot help us see how much of our gene is being expressed, as it codes for every gene in the mosquito. RNA, however, is a direct result of transcription(Going from DNA to RNA) which means that every gene in the RNA is being used in that specific tissue. The genomic DNA must be removed from the RNA sample in order to measure gene expression. This is done in process called "DNase Treatment".
DNase treatment, like many microbiology protocols, involves using a Buffer to set the proper salt concentration and pH for our reaction to take place in. In addition to the buffer, we place an enzyme called DNase into the samples. DNase will eat up all the genomic DNA in our sample. In order for this reaction to take place, the RNA samples need to be in a 37 degree water bath for 30 minutes. After that we add a chemical called "EDTA" to stop the reaction. This is done because DNase will start to destroy the RNA as well if it is left unchecked. Now that the RNA does not have any Genomic DNA in it, DNA can be made from the RNA.
We then perform a process called "Reverse Transcription". We use an enzyme called "Reverse Transcriptase", another buffer(again to set optimal pH and salt concentration), primers(small chains of nucleotides that attach to our RNA allowing DNA to be formed) and dNTPs(Nucleotides that can be used to form complimentary DNA). This reaction takes place in a thermo cycler(named because it cycles the process through several different temperatures). Now that we have cDNA, PCR can be done.
PCR takes the DNA that we have and multiplies it. We don't have a lot of DNA after it is formed from our RNA, so we need to get more. Each strand of DNA that was formed during reverse transcription is replicated many times. Many, many, many times. 2^32 times. After we have plenty of our DNA, we run a gel.
A gel can tell us many things. In my case, it confirmed that I performed the previous steps correctly(resulting in workable DNA). The gels I posted are listed from lowest exposure(top) to highest exposure(bottom). The well farthest to the left is called a ladder. The ladder contains DNA of known lengths so we can compare our DNA to see how large it is. The second well is our NTC(Non-Template-Control). It's essentially GO TAQ(A rare enzyme that can withstand extreme temperatures) and water. Wells 3-6 are DNase treated RNA. We would expect to see nothing in wells 2-6 because they do not have any DNA in them. Wells 7-10 contain our post PCR DNA samples. We used an actin primer to detect how active actin was in my samples. The three wells showed heavy actin activity, while the last well did not. The first three wells are samples from mosquito midguts, ovaries, and fat bodies(respectively). The final well was DNA prepared from mosquito heads. Actin is most commonly found where there is high muscle activity. Because the head has high levels of nerve cells and low levels of muscle cells, this result is acceptable.
I will be practicing these protocols for the rest of the week, and possibly next week as well. I'll have more pictures of some of the lab equipment(such as QIAcube and the Thermo cycler) in my next post!
