Deja Cunningham, Week 3+4, Gathering Data

Week three and four have been very eventful and promising. In the beginning of the third week, I had my mouse lab orientation, which now means that I have granted access to the mouse room by myself! Although it took longer than I expected for me to get this, I am excited that I have my own independence now to visit the mouse room.

Elizabeth sent the lung samples that we collected from the dissections the previous week to the Duke CORE facility to be embedded in paraffin. It takes at least a week to get the sample back so in the mean time we have been working on the ex vivo project involving the bone marrow derived macrophages. Since Sarcoidosis is a chronic inflammatory disease and its pathogenesis is still unknown, we are only using the macrophages to see the granuloma structure under a microscope, if there are increased expression of specific genes during the disease, and how different concentrations of carbon nanotubes affect granuloma growth. Our main gene of focus that week was Osteopontin, which showed increased expression in studies before. To look at the gene expression we use qPCR, which stands for quantitative PCR. Compared to regular PCR, which simply makes ample copies of DNA, quantitative PCR looks at the amount of copies there are of a specific gene in a sample and compares it to the normal levels of a “housekeeper gene”. A housekeeper gene is simply the control and is a gene found in all cell types. For our experiment, we used 18s rRNA, which is the gene for ribosomal RNA. One important thing we had to do before the qPCR was create cDNA using reverse transcriptase. This enzyme converts RNA into DNA. This is essential because we can’t copy RNA since it is very unstable. 

We took the cDNA from six macrophage samples and added it the qPCR mix (H20, Forward primer, Reverse Primer, 2xKappa) already in the 36 wells We added different primers for each of the genes. After that was done, we sealed the tubes and put in in the machine to start. In order to compare the amount of DNA, we look at the number of PCR cycles it takes to reach the threshold cycle (CT), which is the point on the graph right before the exponential phase (when DNA doubling begins). Usually the lower the cycle number, the more DNA there is because it takes less time to reach threshold. We interpret the data from qPCR on excel.

Data from qPCR

Reading the data can sometimes be confusing if you don’t know what everything is. The three numbers in the first row next to the number one represents the CT values from the DNA of sample #1. We use three wells for the same sample as a control to make sure the data is valid and consistent. We take the average from the three wells to use as the main CT value. Then, we find the standard deviation. Next, we find the difference of the CT values for each sample of the housekeeper gene (control) 18s rRNA and the OPN gene (experimental). This is called the DCT. We then find the DDCT, which is the difference of the control DCT and experimental DCT. Finally, 2^-ddct gives us our expression fold change value. We put that data into a graph. Although the data suggests that there is no difference in the Osteopontin levels of the carbon nanotube treated mice compared to the naive mice, this could be because the primers didn’t work. We are looking into doing qPCR again, but for five different genes, which are also said to be increased in Sarcoidosis patients.

Besides qPCR, I did some other cool things in the lab. I learned how to extract bone marrow from mice. Surprisingly it was easier than I expected. All I had to do was cut out the bone and use a syringe filled with PBS to flush out the bone marrow into a plate. I then made a single cell suspension by pippeting the mixture into a tube through a special filter. I also used the cryostat microtome to cut 7μm wide slices of the lungs (image to the right). Elizabeth and I used a high magnification microscope to take pictures of the granuloma structures of the bone marrow derived macrophages. It was really cool. She even taught me how to design primers for three genes we were planning on using in the upcoming qPCRs. 

The last main technique I learned in the lab was ELISA. Because our qPCR results did not prove that the expression of Osteopontin is higher in Sarcoidosis, we wanted to see if the concentration of the protein itself was higher in the supernatant we collected from the macrophages. Usually in ELISA, an antibody is coated at the bottom of a well and the substrate specific to the antibody binds to it. A secondary antibody attaches to the substrate and glows once this interaction occurs. Depending on the color level of the light, it tells us the concentration of the substrate in the well. Our results came out great!! The color of our well was very blue in the samples from the carbon nanotube treated mice compared to the untreated mice. We are very hopeful that we will have good results in the other samples we have in the freezer. 

                                                                                     

I might have had the most fun in terms of activities these two weeks. There were so many things we did and discovered. My mom and I went to a cute burger place on ninth street called Burger Bach, which gets their beef from New Zealand. I mean come on NEW ZEALAND!! Keeley, Shawn, and I went to the bowling alley in town one night to have some fun. Shawn made a bet with Keeley that if he won, she had to buy him beats. Now of course he was going to lose because its Keeley. She actually bowls in her free time (only pros like her have their own bowling ball). Keeley, my mom, and I on the fourth of July weekend walked through the Duke gardens before my dad and sister arrived. The way it looks on a map can be deceiving. It is HUGE, but it is very beautiful. On Sunday we all went to the Raleigh Museum of Natural Science and met Keeley’s friend there. We saw a really cool race exhibit there. AND didn’t I say we would go back to Elon just for the tacos? Well we did that too. We vowed to go at least one more time before we leave. Don't worry we went running this week too to lose all the weight we probably gained from the tacos. 

Top left: Meal from Burger Bach, Top right: Bowling Night, Bottom: Duke Gardens

It’s crazy to think that I only have two weeks left. I’m definitely going to cherish my last moments here. I can’t wait to enjoy my last two weeks here at Duke!