Research Question
Hello again! It's now been two weeks of research, and I have to say that I've learned so much. I didn't even know how to pipette before I got here...now that's a serious learning curve.
Anyway, funnily enough, as I write this, my plates for my initial assay are shaking, and I'll take fluorescence readings on the plate reader afterwards to measure apoptosis, so I have 22 minutes and 20 seconds to finish up here.
To get straight to the point, my research question is basically this: Can an aptamer-siRNA chimera be a functional treatment for cancer? Which is to say, can the chimera first be specific enough to be taken up into the correct cell type, and then is it effective in knocking down the desired cancer gene? The chimera I'm working with (or will eventually be working with) targets prostate cancer specifically, but depending on the results I can achieve, it may be possible to extend some of my findings to other cancers as well.
In order to answer my research question, I first have to do this apoptosis assay I mentioned. The first part of this (which has consumed the better part of the last two weeks, as a matter of fact) is simply trying to get the positive control to work - we're using a known chemotherapy agent and trying to see significant cell death from that, just to make sure the cells we're using will work. Given that this is just the first part of the first phase of my project, I really didn't expect it to be so time-consuming and labor-intensive. I thought we could just plate some cells, throw on some DOX, verify that the cells died, and be good to go. Well, I also mentioned a very high learning curve, as this is my first lab work...
As it turns out, there is a lot to be learned just for this positive control portion of the apoptosis assay - anything from counting and plating cells, to centrifuging/concentrating cell volume, to doing dilutions, and more. Part of the issue is that I am starting from scratch in learning all the techniques, but another part of the issue is that this assay is pretty much new to everyone in the lab. That is, we have no idea what concentrations we need of the chemotherapy agent, nor how long this agent needs to do its job and kill the cells, etc. Everything is a learning process, but each time we run a plate and realize our data isn't quite what we want it to be, we can use that information in a future trial in order to obtain better results. Anyway, the bottom line is that hopefully, once I get this positive control going, I'll then be able to check the siRNA and actually get into a little more of the meat of my project.
But aside from just doing this apoptosis assay, I'm also working on the initial stages of phase two of my project. Phase two consists of actually making the chimera. Once again, even the basics are taking me a long time because I have to learn everything from the beginning, but so far I've done things like digesting a plasmid with selected enzymes, running my RNA fragments in PCR reactions, lots of PCR purification (and I see a lot more in my future - woo hoo), and gel electrophoresis. This phase of my project is a little different because it's not so much results-oriented but rather product-oriented; I'm not looking at numbers or statistics to analyze what I've done, but rather just attempting to get a certain product from the reactions I'm running.
At any rate, it's pretty cool to start seeing how this all begins to fit together. I've also begun keeping a lab notebook, which I'm unexpectedly enamored of - I don't know why, but I just really love the feeling of taping in results of data I've actually worked to collect. Like a nice little version of my eight weeks in the lab this summer, in book form.
I guess I'll be blogging again before too long - it'll be interesting to see what the next couple days bring!
