Week 4 Sabine

Oh my goodness, I am over halfway done with my internship! Where did time go, what is happening? It’s going by so quickly.

This week in the lab, we started off with the Qubit (pronounced cubit). The Qubit is a little machine that tells you how much RNA is in your sample. We already used the QRT-PCR last week to find out how much RNA we had, but we are using our leftover samples to run through the Qubit because it is more accurate. That way, we can compare last week’s results to this week’s results to make sure they look about the same.

Qubit Video

Rob using the Qubit

Before putting the samples in the Qubit, we added some liquids to our samples. These liquids contained things called “fluorescent tags.” These fluorescent tags bind to RNA. That way, the Qubit could measure the fluorescence of each sample, and by measuring the fluorescence, it could determine how much RNA there was.

Once we got the data from the Qubit, Rob showed me how to use Excel to organize the data, do a few calculations, and generate graphs. He did the first graph, and then let me do the rest on my own. It was actually pretty easy, and kind of fun!

The data came out well, and we found out what we needed to find out from that experiment. In an earlier blog, I talked about how there were wildtype, heterozygous, and knockout mice in the experiment. However, I neglected to mention that it was very difficult to “knock out” the entire Sfrp5 gene. Because it is so hard to knock out the whole gene, Rob used knockout mice that still had the first section of the gene intact. The goal of the experiment we were working on was to find out if the first part of the gene was still present in the knockout mice. We were able to collect RNA from Exon 1 (exon 1 is the first “section” of the gene) in the knockout mice. We knew that we collected RNA because the Qubit said that we had RNA in that sample. Therefore, we knew that exon 1 was still expressed in the knockout mice.

Because we found out all that we needed to know from that experiment, Rob moved me on to a second experiment to work on for the remainder of the time. I am working primarily with Rea on this new experiment. This experiment involves a different gene. This gene is called Bmp3, and it inhibits bone growth. In a previous experiment, Rob got some very perplexing results regarding Bmp3, and so he is having Rea and I repeat the experiment, except this time with more mice, so that they can better understand the results.

I will now explain the perplexing results that he got: Rob was studying Bmp3 expression in mice. He recorded Bmp3 expression in fat (specifically, inguinal fat) and liver. These mice had extremely low Bmp3 expression in the liver at 5 days of age and 10 days of age, but then Bmp3 expression skyrocketed once they reached 21 days of age. However, in the inguinal fat, they had very high expression at 5 and 10 days old, and then Bmp3 expression plummeted at 21 days old. So, essentially, Bmp3 was expressed in opposite ways in inguinal fat and liver fat in young, developing mice. In liver, expression was very low and then very high, while in inguinal fat, expression was very high and then very low.

I thought this was pretty fascinating, so I’m kind of excited to see how our results turn out! It’s interesting, because these mice  are young and still developing, so they are currently growing their bones. That means that Bmp3 is very important in this stage of their lives, because Bmp3 is a regulator of bone development. So the expression of Bmp3 in young mice is more interesting than it is in older mice who have already developed their bones.

I’m working with Rea more, now, and we have been isolating RNA. I’ve isolated RNA three or four times, now, so I’m getting more comfortable with it. It’s getting to the point that it’s very repetitive and sort of mind-numbing. But you have to be very aware of what’s going on, because one silly mistake could jeopardize the whole experiment! But I’m more familiar with the procedure now. I still mess up, of course, but I haven’t done anything serious! Just little mistakes! Rea is good at making me feel better about messing up. She emphasizes that she has done this many times, and that I haven’t. But not in an arrogant way, it’s just to make me feel better. What is second nature to her or Rob is completely new to me, and she is aware of that. This makes me a little more comfortable with asking her questions. I’m not as afraid of sounding dumb!

While we were homogenizing tissues (I included a video of tissue homogenizing in a previous post), Rea told me about an experiment that she is doing that I thought was very interesting. I know I can learn a lot from her, so I was grilling her with questions. I don’t know why, but I’m really comfortable asking her questions, which is great. One of the genes the Koza lab is studying is called Mest. Rea heard about a study that found that Mest was highly expressed in human samples of “dormant” uterine cancer. In that study, they performed the RNA isolation procedure that I have been following to isolate RNA from uterine cancers that have been removed from human patients and been stored and frozen so that researchers could study them. Because of this finding, Rea decided to study Mest expression in female mice to see if Mest expression was related to the mouse-equivalent of the menstrual cycle. Right now, she is just using a few mice and seeing if the experiment is even worth pursuing, and trying to see if a relationship even exists. However, if she finds something interesting, she said that the next step would be to see if “knocking out” the Mest gene in female mice changes their cycle pattern at all. She said that the study is very high maintenance because the mice cycle every four days, and so she constantly has to check on them to see what stage of the cycle they are in. I thought this study was very interesting because of its pertinence to uterine cancer. I think it would be interesting to compare the Mest expression in the high-fat mice to the Mest expression in the  uterine cancer. I asked Rea if she would ever consider doing that, and she said that it was possible to see how/if a high-fat diet changed the mice’s cycles, but she said that right now she is only doing small-scale preliminary work to see if the experiment is even worth pursuing, so I’m not sure if she’s actually going to incorporate the high-fat diet into her experiment. I would be very interested, though, if she did, because I’m most interested in the link between diet and chronic disease.

Rea invited me to a lecture during the day yesterday, and I went. I didn’t understand any of it, because the guy giving the lecture was a PhD, presenting to a room full of other PhD’s. It had something to do with the immune system and the cardiovascular system, and that’s about all I could comprehend. PhD’s are very very smart people.

I’ve gone on a few runs with the high school girls’ cross country team, and it’s been a lot of fun! One of the girls is pretty much exactly my same speed, so we always run next to each other and chat. It’s great to have a running buddy! Also, a couple nights ago, I hung out with two of the cross country girls, which was really fun. We went and had dinner at Chipotle, and then they made me watch Mean Girls with them because I had never seen it before! I’ve been very busy, but only because my time has been filled with lots of fun things. I am convinced that I will never get used to the humidity, and I will never get used to the fact that there are no mountains. But I’m having so much fun, and I’m learning so much, and I can’t believe that I’ve already been here for four weeks! Bye for now!