6 weeks into thesis!
We are now 6 weeks into thesis!
It certainly hasn’t felt like 6 weeks. We’re wrapping up a bunch of prerequisites before we start our experimental trials. These include primer efficiency curves, as well as making sure our housekeeping genes are good picks for our chemical doses. We will be comparing expression of housekeeping genes in dosed and non-dosed embryos in order to see if expression really is being changed. As a reminder, we are looking at the expression of six antioxidant genes that have been implicated in the oxidative stress response and with our four chemicals, tBOOH, tBHQ, Diquat, and sulforphane. Lab has been great, breeding our fish is getting easier and faster, and I’ve gotten much more comfortable with the procedures we’ve been doing over the past few weeks. I’m still a little wary of RNA extractions, however I feel confident I can do them. It’s pretty nerve wracking considering that if we do mess up, we have to start all over again. To get a sample at this point, we have to breed for embryos, collect the embryos, dosed them with chemical, and froze the embryos.
After that, we have to extract the RNA from those embryos then finally run qPCR with our housekeeping and antioxidant genes. If you mess up at the RNA extraction phase, you have to breed and dose all over again! So, while it is a bit of pressure, it’s certainly doable. Experimental trials will add an extra step of microinjection of morpholino to knockdown Nfe2, which I will explain further in my next blog post. I’ve also delved into writing my actual thesis, and I have to say I can’t sing the praises of summer research enough. It gave me a huge head start on sources and introductory knowledge, and it’s been nice getting to dive deeper and deeper into the primary literature given that I’ve got a good background of our topic. It only helps that I personally find ROS chemistry pretty fascinating. With October coming to a close, I can’t wait to start experimental trials soon!