Hi there, welcome back to Blog #3!
This week was a very interesting week, to say the least. The luciferase assay that I briefly outlined last week did not turn out as expected — at all. Before we go into that, let me summarize what I tried to accomplish.
As I mentioned last week, transfection is the process of trying to insert nucleic acids (in my case a plasmid) into a eukaryotic cell. To do this, we used an enzymatic reaction to poke holes into kidney cells, which consequently allowed the plasmid to enter the cell. After that, we allow them to recover for 48 hours, then proceed to measure the levels of luciferase in each group of cells.
I wanted to test 2 different versions of the RBM20 activity reporter that I had created. I did not know which one would work more efficiently (or at all), so I wanted to compare how much luciferase was produced by cells with the original plasmid (no interrupting sequence) and my two different reporters. I hoped to see that the cells with the new reporters produce low levels of luciferase relative to the original plasmid (which should produce a lot of luciferase). However, after doing my experiment, I got results that were the complete opposite. The cells that had my new reporter plasmids were producing an amazing amount of luciferase whereas cells with the original plasmid were producing about 100x less luciferase. I have no idea why this is occurring, so I’m in the process of running a reverse transcription reaction, where I use enzymes to convert the mRNA in the cell to DNA, which I can then sequence to find out what is going wrong.
Fortunately, I do have some great news for today’s blog: I have beating cells!!! I’m attaching a small video of them to this blog post. Note: They seem to be beating very slowly, and that’s because I’ve just placed them in a solution containing no glucose. This process, which we call starvation (obviously), is designed to kill any other non-cardiomyocytes in the well so that we can obtain a pure sample of only heart cells.