samhfscience-blog

[samhfscience-blog]

5/13/2017

Right now I am working on our final presentation with MAYA! We are doing a co presentation because our topics are the same except for how we are collecting data. She studied methylation and I studied expression. Hopefully this will make the whole experience much easier and less stressful. 

Time is running out, so I probably won’t be able to run my last plate. Steve said he would do it for me, so that I can have a bit more data to work with for my dissertation and presentation. This will be my last blog post on this page forever. Goodbye! 

[samhfscience-blog]

4/22/2017

My last real time seems to have done well. There are low levels of variation in the curves meaning they can probably be used as real data points. Steve caught me last time filling the 96 well plate with the master mix before putting in the cDNA. Apparently I am not supposed to do that and i’m actually supposed to put in the cDNA first. However, the data still seemed to turn out fine, so no harm done I guess. I’ll try putting in the cDNA first next time and see if it makes any difference. 

I have been out of the lab for a few weeks because of spring break and sickness, but it is good to be back to start collecting more data. I’d like to have some good stuff to present and maybe to compare with Maya who is doing methylation. Our projects are pretty to compare because we are looking at opposite sides of the gene. I am looking at expression and she is looking at methylation. 

[samhfscience-blog]

4/8/2017

So far, all of the real times I have done have not gone well. I am not completely sure what is wrong with them, but the expression curves have pretty wide variability. To be more specific, the third curve is what usually doesn’t fit with the first two. Issues may be that I am not mixing enough or some other procedural problem. I will try doing extra mixing next time around so that I can make sure everything is in solution. 

The end of the year is coming quickly so I need to start collecting lots more data for my final project. I submitted my rough draft, but a lot of work needs to be done before I can present. 

[samhfscience-blog]

3/11/2017

As the year comes to a close, I have to now get started on my final project for my lab work. I have done a lot of research on the topic because of the literature review, but I have not done that much of my own research to discuss. I will likely have to leave a lot of space in my rough raft for my final results. 

Steve just let me know that he has some samples he is preparing to test for maternal immune activation. This is great because the samples I have tested so far have all been controls. It will be very interesting to see how MIA affects oxytocin expression in these mice. On the down side, the samples I tested last week have still not been tested by Steve. It is all kind of a lose-lose situation. If Steve’s tests come out fine then it means I did something wrong, but if they come out inconclusive, then we don’t know what to do about the samples. 

[samhfscience-blog]

2/26/2017

I spent a few hours making a plate to cover the olfactory bulb in several mice with a few different assays. Unfortunately, for an unknown reason, there was a huge deviation with the expression levels between each of the triplicates. I had to re run the samples yesterday and I have not seen the data yet. On the upside, I was able to finish the plate in half the time as it took before. I was home before five, which is extremely quick for my standards. But, if the plate doesn’t turn out well again, then I’ll have to figure out what went wrong and maybe try again. 

I have a rough draft of my dissertation due in a few weeks and I have not started. The issue is that I really don’t have that much data to work with. Only two or three plates have been run and I don’t think any of them are actually testing maternal immune activation, just a bunch of control mice. I’ll figure something out. 

[samhfscience-blog]

2/12/2017

I spent all of last Friday working on a plate for real time PCR. It took a lot longer than Libby expected, plus some mathematical errors, so I ended up staying here pretty late. However, the data ended up looking pretty good, so overall it was a success. I’ll get better at pipetting and sorting out plates and doing the numbers in the future, so I shouldn’t have such an issue with it again. 

I’m not sure what exact next steps are, but I expect I will get some more RNA and go from there, following the same steps as last time, to collect data using real time PCR. 

I am slightly worried that I will not have a lot of data to work with by the end of the year for my presentation because the rate at which this is working out currently is pretty slow. no fault of anyone’s, its just how this research is. 

[samhfscience-blog]

1/29/2017

I spent yesterday doing cDNA generation and attempting to quantify the concentration of RNA in all of our samples. I have been getting widely different data from the single channel nanodrop, the eight channel nanodrop, and the QuBit. Apparently the QuBit needs to be recalibrated, but the nanodrop machines, to me, seem most unreliable because they are giving widely different pieces of data. I finally used some data from the QuBit and single channel nanodrop to normalize the samples to 125microL. the maximum volume was 16microL, but several of the samples were well below 125microL even with 16microL of RNA, so I just used as much as I could. I got half way through putting the enzyme mix into all of the tubes to be put into the PCR machines when I found out there wasn’t enough, so half of my samples are still sitting in the freezer to be generated when a new PCR kit arrives. 

As much as science tries to be exact, there is a lot that can go wrong which makes some of our final results to just be the best we can do.

[samhfscience-blog]

1/15/2017

I ran an optimization plate the Friday before the snow and it didn’t turn out too well. There was an issue with the math before I filled the wells and what made it worse was that I didn’t know I should make a solution of the mastermix and the water in order to make a more accurate plate. Steve says I will be running a new optimization plate and I will use a better method this time around. 

In my last meeting with Simon I asked him about working in the vivarium with the mice. Steve is working on putting me on the protocol and I have done some online training so that I can go into the vivarium and maybe even do some of my own dissections. This is pretty exciting to do, especially at my age. 

I have learned that working in a lab is entirely a group effort. Not just for the people on your team, but with the scientific community as a whole. This includes other research teams, the institution you are working with and organizations like IACUC that keep everything in check. This can be a great thing, but it can also slow things down a lot, especially when certain verification is required to perform certain tasks.

[samhfscience-blog]

12/11/2016

Dear readers,

We finally got some data! Steve and I generated some cDNA and then used qPCR to test the expression levels of several genes. There were four genes that relate to Oxytocin and two androgynous controls. The data was not totally surprising. For example, OXTR expression was very low in the liver, as to be expected. We will be testing more mice in the future once Steve dissects a few more mice. Steve is currently trying to figure out what I can do in terms of dealing with actual mice in the vivarium because I am only 17 years old. 

In the future we will be testing more controls and doing so until our Maternal Immune Activation testing is approved. These mice will be assaulted with Poly(I:C) to induce immune activation without actually giving them a disease. Once that happens, we will be able to compare data from the control groups and those we hypothesize to have a decreased OXT expression. 

[samhfscience-blog]

11/21/2016

My project is getting off to a good start. The samples collected in the last post were extracted for their RNA. I took the RNA samples and synthesized cDNA, or DNA copies, to use in the ViiA machine to collect some data on OXT extractions. The rest of the year will likely look similar to this, but hopefully done with a bit more efficiency. I will start to collect data on more samples that are collected under different circumstances to test OXT expression and hopefully get a good idea of how maternal immune activation affects it.

I turned in my literature review last week which covered several papers about OXT, MIA, and ASD. There was one interesting article about how OXT is used in bonding between animals, especially in mother to infant relationships. This paper didn’t have anything to do with MIA or ASD, but it showed how OXT has an affect on the brain. Most of the papers that covered MIA explained how there is overwhelming evidence to support a relationship between ASD and MIA. I am still a bit confused, however, about how OXT specifically affects ASD. I know that it has certain anti inflammatory affects with microglial cells and it plays a part in things like the GABA system. OXT is so complex in how it affects different systems and, ultimately, the etiology of ASD. It is an interesting topic and I want to learn more about it. 

[samhfscience-blog]

11/4/2016

Steve collected several more samples, adding up to seven(?) mice so far. I tested the mRNA from the first mouse that Steve extracted in order to see the expression levels for several different concentrations. The organs that Steve extracted most recently can soon be used to continue with my project. They are being stored in an RNA Later solution, which works as a preservative. 

I just found out that I have a literature review due on the 14th of November, so I have started taking notes on some of the papers that Steve has sent to me. The most recent one I read was a study to test the effects of insulting an embryo with poly (I:C) in different stages of development. For example: the study found that GABAergic cell development was altered due to changes in the methylation of the Lhx and Dlx genes when exposed to poly(I:C) in late gestation (GD 17). The GABA system works to inhibit neural excitability, which, when gone wrong, can lead to psychiatric disorders such as schizophrenia and autism. The interesting thing is that even in middle gestation maternal immune activation (GD 9), where there are no “overt morphological abnormalities,” there are still disruptions in GABAergic neurotransmission. 

[samhfscience-blog]

10/21/2016

What have I been up to in the lab? 

Again, not much, but Steve showed me the samples he collected from the mice the other day. He showed me a few pieces of a mouse brain, a uterus(?), and part of the liver. The uterus and brain will be the test groups while the liver will be sort of like a control since its cells don’t change much due to environmental stressors. 

We are waiting for some EDTA and HEPES to arrive, so we can preserve the samples for future use. EDTA, or Ethylenediaminetetraacetic acid, is more than just a long word. EDTA does something in the solution that reduces the reactivity of metal ions, which allows it to be used as a preservative. HEPES is a buffer, one which I have used before, which will keep the pH stable after the EDTA has been added. 

The brain samples have been put through some type of sonication process, so it is now a brain soup, if you will. A sonicator uses sound waves to break apart materials in a solution. I have used one before to dissociate ZnO2 into solution, but it didn’t work because of how hydrophobic the compound is. 

[samhfscience-blog]

10/7/2016

I have spent the past few weeks in the lab reading articles about what I will soon be researching. I have learned about how Oxycontin affects social tendencies of mammals. Voles, for example, are monogamous and often will connect with adolescents that are not theirs. This is known as alloparental care, a trait also seen in humans. This shows Oxycontin’s effects on affiliative behaviors.

Another paper I read was about the correlation between certain environmental factors in pregnant women and autism in the babies. Maternal Immune Activation, or MIA, is caused by immune responses in pregnant women. In a specific study, pregnant rodents were injected with Poly I:C, a molecule that triggers MIA. The offspring were found to display traits that are present in Autism Spectrum Disorder, or ASD.

[samhfscience-blog]

9/23/2016

My first several weeks in the lab have been, honestly, been pretty uneventful. But I have not given up hope. I know that in the next few weeks, things will start to pick up. My goal for finding my first laboratory job was to learn about what lab work is like. My goal for this year is to gain a more in depth understanding on slightly more independent research as well as learn about an entirely different field of study. Since I do not know exactly what I want to study in college, this will help me narrow down my interests and, hopefully, get into college as well.

Next week I am told that I will begin to start on the ViiA PCR system to look at mouse tissues.