Week 7- Taking and Making Genetic Material

Anita M -

Hello! In my last post, I mentioned treating my cells with tunicamycin (TM) to stress the cells, emulating the effects of a heart attack. The TM treatment worked well and we didn’t experience a significant amount of cell death which was good!
Below are pictures of my cells. The picture on the left is cells without the TM treatment and the right picture is cells with the TM treatment. As you can see, the TM-treated cells look stretched out and almost web-like in comparison, indicating high levels of stress.

We left the TM on for 24 hours and extracted mRNA from them the following day.

As a quick reminder, DNA (our genetic material) is used to make proteins within our body. But for DNA to do that it has to use messenger RNA (mRNA).

DNA –> mRNA –> Protein

What we did in this project was insert a certain kind of DNA in our cells to make a particular protein (GRASP55). Now, we want to see if that inserted DNA also caused certain other proteins to be made. We can observe that by looking for specific mRNA in the cells.

So, we extracted the mRNA. But RNA is really unstable, so we had to turn it into complimentary DNA (cDNA). Currently, my cDNA is sitting in a freezer waiting to be analyzed, which is what I’ll be doing this week!
Using a process called qPCR, we are going looking for changes in the mRNA of 4 genes: ATF6, GRP78, GRP94, and ANP. If there is any change in the levels of mRNA of these genes, we can hypothesize there is potential link between GRASP55 and the genes!

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    kira_a
    This is super interesting as always, Anita. I am glad to see that your cells are being more cooperative this week! Could you explain the process of qPCR a bit more and how you will be using it to look for changes in the mRNA of the genes you are interested in?
    March 27, 2025 at 11:32 am - Reply
    camille_bennett
    Hi Anita, so fascinating. Did you notice any other changes in the TM-treated cells besides the stretched, web-like appearance?
    April 1, 2025 at 10:22 am - Reply
    anita_m
    Hey Kira, qPCR is how we can measure gene expression. We do this by extracting RNA from cells (since that RNA contains the transcript of the genes that were expressed) and then we create cDNA from that RNA. We put that cDNA into a qPCR machine and essentially measure the speed of replication by measuring the amount of "cycles" the machine goes through. We then compare the gene of interest's number of cycles to the control gene's number of cycles and measure the difference between the two. For my experiment, we used the qPCR to compare our genes of interests and measure the gene expressions of ATF6 and GRP78 -- two genes that are typically activated during cell stress.
    April 1, 2025 at 11:32 pm - Reply
    anita_m
    Hi Ms. Bennett, Thanks for asking! There isn't really much we could judge the cells on at this stage except their appearance under the microscope. We did notice that there were large gaps between cells, but that is characteristic of stressed cells. Also those large gaps are most likely also due to some cell death, but again, that is a very normal thing to see in stressed cells.
    April 1, 2025 at 11:38 pm - Reply

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