We are quickly approaching the end of thesis!
That being said I know everyone in the lab still has plenty of work still to do and has been working hard. Since my last post, I have been able to expand on my lab skills and process some data that I have been collecting. I am completely finished with my control morpholino diquat exposure trials and have moved on to my nfe2 morpholino trials. I only have one more trial to go until I am to compare the two groups. I will be comparing the distance between otoliths, the width, and length of the otic vesicle using stereoscope microscopy. Additionally, I will get to compare neuromast development using confocal microscopy.
I have been extremely busy with the qPCR aspect of my project. qPCR, also know as Quantitative PCR, is a technique that we use to allow us to determine the change in relative expression of a particular gene. In order to do this, we extracted RNA from AB and nfe2 knockout 96hpf fish that were either treated with water (control), tBOOH, or diquat. We then perform a cDNA isolation, to obtain the cDNA that we use for qPCR. I originally selected six genes to test oc90, sparc, otop1, otomp, otolin1-a, and stm. Early on, I found that otlin1-a was having little to no expression at 96hpf. All other genes were showing expression at 96 hpf. The only significant change that we found for out AB 96 hpf zebrafish was down-regulation of the gene oc90 in the diquat treatment group. As for out nfe2 KO zebrafish, we found a significant down-regulation of sparc in the tBOOH treatment group compared to the control and diquat. We also found an up-regulation of the gene stm in the diquat treatment group compared to the control and tBOOH.
The last few weeks will be spent not only analyzing and collecting the rest of my data but also to try to offer an explanation for why these particular things are happening. For me, this is my favorite part of science! Understanding what your data actually means allows you to gain further insight into the broader implications of your study!