Amber #Entry 3 Moving into vitro!

   The previous week has been both disappointing and exciting. After gaining success in the previous experiment, we confirm that DVG mutant 5P5X3P15 (longer oligonucleotide on the 3 prime of RNA) showed great immunistimulatory. Out next step is to show the consistency of such phenomenon by repeating the experiment for three times and adding more mutants to test the position of 3 prime nucleotide on the antiviral response. For the current experiment, we set up 24 samples, including positive and negative control.  However, the experiment failed as the GAPDH gene failed to show expression. GAPDH is a housekeeping gene (we added as a primer to qPCR) that functions as to evaluate the success of the whole experiment: if GAPDH doesn't express in the cells, it means the experiment fails for two potential reasons. One is the cells were alive before ABRT (reverse transcriptase), but the ABRT failed to make DNA on the template of RNA. The other reason is that the cells are stressed or commit apoptosis before its immune system can response to the viral molecules. After the troubleshooting ,we redid the experiment again bu the qPCR results weren't that optimal either even the GAPDH gene expression was good. In order to the find the problem, we talked to our PI and we concluded that maybe the cell lines LLCMK2 doesn't work. I was first frustrated because all the work put into the experiment turned avail . But my mentor said in science, sometimes to prove something is wrong is just as important as to prove a right thing.
  On the other side, the postdocs were trying to push the experiment in vitro using mice. I didn't get trained to work directly with mice, so I was standing aside and watching them do the whole experiment. At first they need to shake the flank of the mice because that's where the tissues are most concentrated. Then they inject the same mutants with dye into the flank and place the mice for a day to observe post-infection for 24 hours. The mice were placed to death after the experiment (poor mice sacrifice for science ). After extracting the tissues with dye the next day, the same steps were required to do the full analysis to examine the immunostimulatory (no transinfection since its in vivo already, and RNA extraction, ABRT, qPCR etc...). The RNA extraction was a bit different because at fist the tissue with trizol need to be processed with homogenizer to homogenize the tissue with trizol at the maximum speed for five times. After this, the process followed was basically the same except it required a few more centrifuges to get rid of any residues. The qPCR results turned out to be great. The success of vivo system revealed the fact that there must be a parallel cell lines in vitro system that confirmed its activity in vivo.
  The success in vivo system was great news because it means that there is still chance in finding the right motif with different cell lines. Our next step is to try the U937 suspension cell lines for pilot experiment and rerun the experiment again. Moreover, the U937 suspesions cells I cultured last Sunday survived!! (yayyy my first success in cell culture technique).
  Emma and I went to Chicago for the weekend and visited Uchicago. Unfortunately we spent fourth of July on plane...... Anyway Chicago was amazing and food was great (deep dish and Garret popcorn). I should probably end here before this scientific blog turns into a food blog.
   Two more weeks left in Philly!