Lab and fish work begins!
We began this week in the Williams lab by testing the efficiency of our primers.
We did this by diluting cDNA, which we had previously prepared, by powers of ten to have 1:1, 1:10, 1:100, and 1:1000 concentrations. We ran qPCR with the primers we found to work last week hoping to get higher crossing threshold values for the more diluted cDNA. By doing this, we can ensure that the primers accurately correlate to the amount of cDNA present in a sample. Overall, we were fairly successful and found our primers to be efficient, although we had a couple dilutions that had to be repeated. These experiments were more preliminary work that we had to get done in order to be able to begin changing variables and testing the effects of our chemicals and Nfe2 knockdown.
Later this week we got into some more exciting stuff- our first breeding of the year! This was the first time breeding for some of us and the first time since July for others. It was a learning experience in patience and perseverance. Somehow the fish seems to swim faster each time we breed. Being one of the more experienced zebrafish breeders, I was able to teach the students who had never set up breeding/collect embryos before. Talking about the process and explaining it was a good review for me as well to remember not only what we do in what order, but the reasons behind everything we do. We set up six tanks, each with two males and two females. Sadly, only two tanks ended up producing embryos, but it was good practice for both us and the zebrafish. We did get enough embryos to begin running some experiments to find the best housekeeping gene. To find the best housekeeping gene, we’re testing three genes that have previously been identified to be stable across development and chemical exposures. The three genes to be tested are 18s, elfa, and β-catenin. We will use our primers for these three genes on samples of embryos dosed with one of our chemicals (TBOOH, TBHQ, or diquat) at each of our time points (2 hpf, 48 hpf, and 96 hpf) to determine which is the most stable across treatments. We’ll hopefully finish that up next week.
Every day we get closer to beginning our experiments on Nfe2 knockdown embryos, and with every preliminary step thesis feels more real and more exciting. I can’t wait to get going with morpholino injections in just a few weeks!