Week 5: More Fireflies!

Mar 24, 2019

Hello! Welcome back to Blog #5!

This week I solidified my plan for the coming few weeks, which gave me some peace of mind in terms of certainty about what I’m doing next. After doing some online research, I found that my initial assumption with my luciferase assay was wrong. Normally, only cardiomyocytes express RBM20, so I used HEK293T cells (kidney tumor cells) to test my splicing reporter. However, the key word in the previous sentence is “tumor.” HEK293T cells are tumor cells, and though normal kidney cells shouldn’t express RBM20, these cells do, which is why I didn’t see a difference in luciferase production when I added RBM20 to the cells. I confirmed the data I found online through a simple RT-PCR, where I use RNA extracted from HEK293T cells to show that RBM20 is expressed.

Now, my plan for next week is to run the same assay as before; however, I will use siRNAs to silence the expression of RBM20 in the HEK cells. This way, there is no endogenous RBM20 expression that can act as a confounding variable in my assay. siRNA stands for small interfering RNA, which silence the expression of a certain gene by binding to its mRNA transcript. This binding inhibits translation and thus is a useful tool in selectively knocking down the expression of specific genes in a cell.

2 Replies to “Week 5: More Fireflies!”

  1. Pranavamshiva V. says:

    So when I first started out at my lab, my professor told me to practice using HEK293T cells, because they were really good “reporter cells” as he phrased it. They’re convenient, pretty loosely adherent, and they give you pretty good information as far as RNA and proteins are concerned. Also, it’s good that you know what you’re going to do for the next few weeks. Hope everything works out!!

  2. anuradhamurthy says:

    How do you titrate the amount of siRNA required to completely inhibit the expression of endogenous RBM20? Also how stable is RBM20? Will it last from parent cell that may have it and be distributed to daughter cells?

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