Amber #Entry 4 Getting the results!

Hellos,
   This is my second to the last week here and I am getting more and more independent on my project. This week I ran the whole experiment by myself from RNA extraction to qPCR. This was my first time doing the whole experiment and the results of the first repeat didn't go that well.
   As you can see in the picture, the wave length didn't occur until the very late cycle, which indicates that the signals didn't get amplified that well. Moreover, the CT value of GAPDH (a housekeeping gene, existing in every cell, used as a positive control of the experiment) was high, which means that the the data from the experiment cannot be used. In the matrix using to calculate the copy numbers of genes expressed to each primer, the value of GAPDH is the only variable used in the calculation, if the value of GAPDH is too high the whole copy numbers will be high and the whole data cannot be used to determine the immunostimulatory response.

The qPCR amplification plots.


 

There are several reasons for the failure of GAPDH. One is the cells are too distressed by the virus and they might be dead by the time of the qPCR. Since the transinfection took place a few days ago, this cause wasn't considered. Another possible reasons might be the failure of abRT. If the RNA wasn't fully copied into cDNA, the qPCR might fail. Therefore, I talked to the postdoc the next day and designed a troubleshooting experiment. I took two old abRT samples and the same RNA samples for new abRT. These four are used to test my abRT. I also chose two positive controls to test my qPCR. I checked the results today and the one with new abRT worked yayy. 

   

Harvesting the virus from chicken eggs. 

    Besides my main project, I was also helping other postdocs with their project. One of the most common way to clone virus to make multiple copies of Sev virus is to inject the virus in the chicken egg and harvest 11 days post infection. 11 days is crucial because the egg doesn't launch immuno response to kill the virus and the egg won't be dead because the virus is too strong. Basically we took out the liquid from albumen while avoid getting any yolk and blood in it. I think this is really cool because this is my first time seeing it:))