More recently in the lab, my time has been focused on optimizing and performing the reactive oxidative species (ROS) assay. As my lab team has not used this particular assay before, one could say that I was breaking new ground with this experiment in the lab. We thought it best to establish and perform the controls before beginning the actual experimentation.
We know that chemical hydrogen peroxide will produce ROS in cells no matter what. After determining the proper concentration range to use, experimentation was just a matter of seeding the cells into a 96-well plate overnight, giving the cells treatment and performing the assay the following day.
The first trial wasn’t completely unsuccessful; however, something was clearly amiss. After communicating with a Cell-Biolabs (company the ROS reagents are from) technician, we learned where we went wrong. In the end, the main problem came down to two very fixable issues: 1. We hadn’t seeded enough cells in the plate the night before and 2. We needed to change to a specialized 96-well plate that would make the experiment easier and give us more data points-which the experimental protocol did not specify in its instructions. Believe me, I wasn’t happy that the company hadn’t included this key information.
After making these two simple adjustments, we were able to properly establish our positive control and were also able to obtain over 200 working data points for each sample. In the final portions of experimentation, we will actually be able to treat the cells with cytokines (chemicals that simulate cancer type environment by creating replicative stress) and determine whether or not there is an ROS response and establish if any correlation exists between ROS and the amount of polymerase eta protein in the cells.
I’ve learned extensive information about modern biochemistry and molecular biology experimental techniques and have become proficient in essentially all of the ones I’ve been taught. Learning how to culture human cells, chemically treat them, count them, and perform ROS assays using them are all things I may not have experienced had it not been for the Honors Enrichment Award. I want to thank the RMU honors program, Professor Harold, Professor VanDieren, and Lindsey Sobolosky for their support and providing me the opportunity to better my academic and professional career. And it is my hope that my experience will demonstrate that you don’t need to go to a huge state school in order to have awesome opportunities as an undergrad, you just need to go to RMU.
Damian Di Florio