Weeks 6 and 7 have been great! I made a ton of progress in the lab, and I got to do a TON of practical procedures. I am excited about weeks 8 and 9 now.
The highlight of these weeks was learning a new procedure - RNA Isolation by Trizol Extraction and Isopropanol Precipitation. A mouthful, but for anyone who wants the TL;DR version, it means extracting genetic material from samples of honey bee brains. DNA and RNA are both extracted from the procedure, and the lab then specifically isolates the RNA (Why do we only care about the RNA? I still am not entirely sure).
Why do we care about this procedure? On the deliverable poster we submitted to the 2017 Arizona Imaging and Microanalysis Society's Microscopy Conference, there was a section mentioning levels of RNA. The lab was conditioning bees with the PER (Proboscis Extension Reflex), and isolating the RNA sequence used to make the GABAa receptor from these conditioned bees, in order to make correlations with the observed behavior and the amount of the RNA sequence isolated. So the lab was trying to find out if high or low levels of the GABAa receptor RNA code could be correlated to the behavior observed from conditioning trials.
This avenue of our research is still ongoing, and I probably will be doing this for the GABAa receptor over the next few weeks. These last two weeks, however, I did this procedure for a different receptor, the tyramine receptor. It's the same procedure, just a different receptor in question. It wasn't my project - I was assisting the graduate student, Mary Peterson, in her research for her Master's degree thesis. Dr. Sinakevitch had me do this to give me the experience I need to have this procedure down. After having me practice micropipetting for a morning, I was ready to go.
Molecular biology requires much precision to get truly accurate results - even a microliter makes all the difference. I had to be really careful the entire time while I was doing the procedure. The micropipetting practice definitely helped, but I had to be careful of a ton of other things too, notably, contamination of the samples and timing for steps such as centrifuging. I thought for sure I was going to mess up something along the way. But I can surprise even myself - the start of Week 7, I did the entire first part of the procedure alone for a dozen samples, no errors.
I was proud of myself - the only real practice I had had was doing some steps of the procedure alongside Mary and Dr. Sinakevitch in Week 6. But the real fun started after I did the procedure alone.
Dr. Sinakevitch showed me what happens next in the second half of Week 7. It turns out that what I had done so far was the first big part of a larger process. I had extracted both DNA and RNA, but I had to separate the RNA. She showed me how to use the kits for separating the RNA, and soon, we were ready for the final part. It was a good thing I did the first part really well - it messes up everything afterwards if you mess up there.
With our small amounts of RNA, we can't really make any conclusions. We needed to amplify our samples. That's what the last part, RTq-PCR, is for. Reverse-transcription Quantitative Polymerase Chain Reaction. This takes the RNA, builds complementary DNA strands, and then amplifies the DNA built, which will show the amount of RNA we got. I'm sure there are more important details associated with this procedure, but I haven't done this last part yet - I only watched Dr. Sinakevitch do it. She said I'd be doing this soon, though, so once I do it, I can get back to all of you on this.
My thoughts on these last few weeks? Amazing. It's getting difficult these days to keep track of everything I know - I welcome the challenge though. This is more biology then I've ever been exposed to. By the time this project is over, I'll be ready to continue doing more of this in a college environment. Also, after doing the RTq-PCR, Dr. Sinakevitch told me the samples I did were excellent, so now, I am very confident going into weeks 8 and 9. I'm just hoping I get to do the RTq-PCR procedure in the next two weeks - it looks really exciting.
Also, I just realized I should post some pics of what's been going on. I'll do that this weekend - the visuals of the last few weeks are too good not to share.
Hey Jaeyoung!
ReplyDeleteAs I was reading about your presentation and poster submission, I was wondering about your interest in science. Does it lie more in hardcore research, or are you also drawn to the presentation, communication, and education parts that you've been exposed to in the symposium and modeled by your mentor?
Unfortunately, I didn't get to actually present (college visit that day), but I do think both are equally important.
ReplyDeleteBy college visit, I mean I went to a college for an interview, haha
ReplyDeleteHey Jaeyoung,
ReplyDeleteYou mention practicing micropipetting before you were able to do anything else. Do you think the week we spent in Dr. Nadja Anderson's lab during our time at KEYS helped you at all with your micropipetting? Additionally, I do not believe we learned about RTq-PCR in AP Bio or during our time at KEYS. However, we did get to practice doing PCR in our initial week at KEYS. How does the process of performing RTq-PCR compare to regular PCR?
Thank you!
Hi Nikash!
ReplyDeleteThe micropipetting at KEYS works the same as micropipetting here, but the practice was still really useful, because I couldn't afford to make any errors, and the amounts I was working with were sometimes really small (down to 20 microliters, for example). Additionally, at the beginning, I was kind of rusty from not having micropipetted for a while.
I can tell you more about RTq-PCR once I get to do the entire process myself. However, I will say it's different because you start with RNA and not DNA, and therefore, the process of extracting RNA beforehand is very different - different chemicals, different timings for centrifuging, etc.
Hey Jaeyoung--I know you are keeping busy, but did you see the announcement about the bee species that officially made the endangered list! Here's the link http://www.usatoday.com/story/tech/sciencefair/2017/01/10/bumble-bee-endangered-species/96394450/
ReplyDeleteIt keep thinking about your work someday helping solve these kinds of problems.
This is an interesting article Ms. Gathas! Yes - the Smith Lab could definitely work on such issues. I know other labs next to ours also work with social insects, so there's a lot of places to work on issues like these.
DeleteGreat work, Jaeyoung! RTq-PCR was a new technique that I learned as well! But it makes sense that you can make copies of the RNA nucleotides to produce DNA nucleotides. Any idea why you would make DNA instead of just amplifying the RNA that you have?
ReplyDeleteI think it's because RNA is an unstable molecule, being single-stranded. Dr. Sinakevitch mentioned this today.
ReplyDeleteThis explanation may also help: https://www.researchgate.net/post/Taq_polymerase_cant_recognize_uracil_in_a_sequence_of_RNA_Is_it_therefore_impossible_that_Taq_polymerase_could_amplify_from_a_RNA_sequence
ReplyDeleteIt states that Taq polymerase doesn't recognize uracil, so I guess that implies there is no enzyme we can use that amplifies RNA