Hey there! Welcome to Blog #6 (we’re more than halfway through!)
I completed my Luciferase assay from last week, which involved silencing endogenous RBM20 expression in HEK293T cells to test my splicing reporter. Essentially, my experiment showed that in cells with RBM20 expressed, twice as much luciferase was produced when compared to cells where RBM20 expression has been silenced. This is great news, and it’s exactly what I wanted to show. However, the experiment I just performed had only 2 data points, since I wanted an affirmation before proceeding with a large scale experiment with 100s of data points that uses a lot of expensive resources (and precious cells).
This week, I plan to perform my luciferase assay with HEK293T cells in a 96 or 384 well plate (i haven’t decided yet). I also want to perform this assay in cardiomyocytes that I’ve been differentiating. Instead of using siRNA to silence RBM20 expression in my cells, I have 2 different types of cardiomyocytes:
- Wild type CM (meaning they have functional RBM20)
- Mutant CM (meaning they have diseased/dysfunctional RBM20)
Doing this assay with cardiomyocytes that are wild-type and diseased is the exact situation in which my splicing reporter will be used in the future, so this experiment will be the final and most accurate test for my reporter in some sense.
For my video series, I’ve begun making a script and compiling statistics on my first video’s disease and I’ll begin filming/animating by the end of the week!