There are so many aspects of research that I never knew about. Every little step requires a very detailed thought out plan that must be performed meticulously. I was surprised by how long it took for me to actually get involved in my experiment. There were many precursor steps I needed to perform in order to execute my final experiment.
First, I had to learn about my project and basic lab use. After observing for a few days, I began to grow my own cells. I had to feed them every day, even on the weekends. These cells soon became like my babies. After nine days I removed them from the plates and isolated their RNA. I had been practicing RNA isolations since the first week, so I was pretty confident. However, I did not obtain nearly enough RNA from the cells as I needed for my next step. I was pretty disappointed that I had failed my babies and that they were basically just trash. I started growing new muscle bundles, and continued to practice RNA isolations. I kept getting really low concentrations of RNA, and I couldn’t figure out why there was a sudden drop from my first few practices. It was pretty frustrating, but after some help from my graduate mentor, I realized I had been using the wrong RNA isolation kit that did not have all the correct reagents added yet. I had basically spent hours adding different reagents that were doing nothing to isolate the RNA. Once I had the right kit, I started getting really high concentration of RNA and good purity. I was then able to run my first RT-qPCR, which amplifies a region on DNA specified by primers. I am using a one-step PCR kit that first transcribes cDNA from the RNA and then amplifies the cDNA. For my first PCR I used untreated muscle bundles just to test if my primers were amplifying the correct genes.
I then had to determine a housekeeping gene that is not affected by the addition of TNF-alpha so that I could use it as a reference for the genes of interest. I completed these preliminary steps and was able to run my first full experiment this week. Fortunately, my hard work paid off. The results seem to be what I was expecting. Now I just have to repeat this experiment 4 more times with different replicates and I will have all my results! It is a really good feeling knowing that all the time and effort pays off in the end. I can’t wait to have all my data so I can begin analyzing and drawing conclusions!
