Spending the last 10 weeks in the Lemke Lab has enabled me to learn a great deal. In the beginning I spent a lot of time training and watching how to complete tasks. As the summer progressed I became more and more able to do things on my own. I definitely still asked a lot of questions because I wanted to make sure I did everything correctly. While precision is very important in science, I also learned that perfection can be the enemy of good enough. For example, when working with some sample I might spend too much time making sure everything looks perfect and the samples might dry out as a result. However, many of the techniques I performed numerous times and each time I got better at balancing perfection with efficiency. I was also able to focus less on the individual steps and more on the overall picture of the experiment.
I spent most of my time over the past 10 weeks isolating RNA, converting it to cDNA in order to use it in a qPCR. The isolation step I quickly improved my techniques, set up and efficiency. After analyzing my RNA I also discovered some of my samples had degraded and therefore learned the hard way which steps I could leave the RNA overnight at and which I could not. When converting the RNA to cDNA through reverse transcription, I had to understand what was happening in the reaction in order to know which reagent to put in at the right step (once by accident I added dNTP instead of oligo(dT) to RNA, which meant there were just a bunch of extra nucleic acids floating around instead of the primer which targets poly(A)+RNA). Loading the 384-well plates for the qPCR taught me attention to detail more than anything else.
Pipetting very small volumes of different reagents into every single well can be difficult to keep track of. In the last couple weeks, my work has shifted more to analysis of all the data that this process produced. It was only with help that I was able to do all the averages, standards deviations and normalizations, making me realize that I should probably take statistics at some point in my college career. This enabled me to work with a software application called Prism, which takes data after it has been analyzed and creates very good and informative graphs.
I’m not sure if this summer has altered my view necessarily of the STEM fields. If anything though, it has strengthened my belief that STEM fields are integral to our country and future. The work of other scientists across the globe continues to emphasize this point. Perhaps the most dramatic example from this summer would be the scientists you have created an Ebola vaccine that efficiently functions in a monkey model. Scientists have the ability to save and better the lives of humanity through strengthening bodies and the Earth.
The program here at the Salk Institute is an excellent example of just that. It enables researchers to interact with their community and show young people exactly what they are doing, why they are doing it and most importantly why the students might like to do it too. I hope that other research institutes around the country are able to start similar programs across the country. In certain areas, like here in San Diego, it seems extremely feasible that if all the researchers took one day out of the year to visit a classroom that most of the classrooms in the county would be better exposed to science.
In the future, I plan to build upon what I have learned this summer by applying the techniques I have learned to different questions and areas of research. Having this experience will show other principal investigators that I am capable of learning new techniques and effectively analyzing the data that they produce. Hopefully, I will eventually gain even more autonomy in the lab so that by graduate school I will feed confident in conducting my own research. I also help to be able to always remember the education program here at Salk so that wherever I may end up working I can bring that idea with me.
Since my last progress report, I have learned a great deal and become more self-sufficient. I have achieved relative independence in the mouse room. This means that I can wean mice (i.e. separate the pups from the mother while simultaneously separating the boys from the girls once they are about twenty-eight days old) by myself and maintain records of what I do. This has also made me realize that maintaining a healthy and well-documented mouse colony is vital to most of the science we do. I have also largely accomplished both my goals.
While I have not completed the entire qPCR process from start to finish (my first goal), I have completed every part of it separately. I am currently still in the process of isolating the RNA because it took longer than anticipated. This is in large part because of the quantity of samples I am using; I am isolating RNA from the spleens, spinal cords and brains of 12 different mice with varying genotypes. As mentioned in my first report, once I have completed the isolation I will convert the RNA to cDNA and run a qPCR to determine expression levels. In regards to my second goal (completing an immunohistochemistry), I have accomplished it in full.
An immunohistochemistry is a technique that researchers use to stain different parts of tissue or types of cells differently. For example, we use it to stain and visualize different components, cell types or proteins in brain tissue. We stain microglia in one color, apoptotic cells in another and so on, using a microscope to see each staining. The staining is achieved by using two sets of antibodies. The first set of antibodies, the primary antibodies, is designed to recognize and bind to antigens of specific elements, i.e. microglia or apoptotic cells. The second set of antibodies, the secondary antibodies, is designed or chosen so that they will recognize and bind to only one of the types of antibodies from the first set. The secondary antibodies are also coupled with a specific fluorophore such that when a specific wavelength of light is shined on the sample, it will fluoresce as a result. Fluorescence is when a photon of light hits a sample and is emitted at a longer wavelength, for example, when blue light is shined on a sample and green is emitted. So, in this way, each individual element will emit a different wavelength based on the fluorophore and the antibodies used.
My primary goals for the next couple of weeks are improving on and perfecting what I have already learned. For example, I would like to improve the efficiency with which I isolate RNA. The homogenization of the tissue in particular is difficult and something I can work on. I would also like to be able to use theconfocal microscope independently because I am currently learning how to work it. It is a delicate piece of equipment that you have to handle carefully. My final goal is to be able to collect the brain, spinal cord and spleen from mice while maintaining the integrity of the organs. This is important so that when we use them downstream for various applications we can get accurate results.
I began my internship at the Salk Institute for Biological Studies in Dr. Greg Lemke’s lab on May 27th. I am working under the supervision of a postdoctoral fellow named Lawrence Fourgeaud. I spent the first couple of days getting oriented and settling in. In my training, we reviewed how to work safely and conscientiously in a lab. I then learned how to handle and work with mice ethically and effectively through hands-on instruction and an online training course. As I finished up my training at the end of the first week, I began doing lab work. One of the first experiments I did was run a qPCR. qPCR is a method to look at the level of expression of specific genes in cells and tissues.
Another technology I learned about is in vivo two-photon microscopy. We are working in collaboration with a postdoctoral researcher in another lab here at Salk (Nimmerjahn lab). We want to image microglia cells in vivo (ie. in an anesthetized animal) using this technique.