It’s been a busy winter!
As Nick mentioned, we are in for busy times with March approaching.
Fortunately, Stratagene sent us a loaner qPCR machine that we have been using in place of the one that was giving us unpredictable results past column 7 on our 96 well plate. I am happy to report that I’ve never been more confident at pipetting my qPCR wells. I haven’t had my qPCR plates fail due to pipetting errors in a long time, and I’m glad I can go into finishing the rest of my qPCR runs with a positive mindset. As we begin to analyze our data, we need to pay attention to our housekeeping genes. As a reminder, these are genes that we will be normalizing our samples to in order to determine relative fold change. Good housekeeping genes are those that are consistent across samples. For our purposes, we need housekeeping genes that will yield crossing threshold (Ct) values that are relatively consistent across treatment and time, which has proven to be a challenging task. The catch is that while one housekeeping gene is consistent across time points, it isn’t consistent across treatment and vice versa. Lucky for us, Larissa and a colleague found a program that can utilize three housekeeping genes for normalization purposes. In a sense, this dilutes the variability we see from one housekeeping gene across all three genes. Using Ct values from all three genes, a normalization factor is calculated. Ct values from our oxidative stress samples are then compared to the normalization factor of all three housekeeping genes. This approach has been cited as a more accurate way of normalizing sample results, given that it is very difficult to find a single housekeeping gene that is consistent across a variety of conditions (time, treatment, etc).
I’m very excited to be able to have analyzable results within a few days, and I’m ecstatic about having gained so much confidence over techniques I used to struggle with. In March, we’ll be writing a lot more for thesis and knocking out the rest of our qPCR runs in order to move on to experiments involving Nfe2 manipulation.