Hi there! We believe that a western blot or immunofluorescence will tell us more about the protein formation that occurs with the expression of the gene (and its mRNA) and where this activity is localized.
Hello Maria! Hydroxyurea (HU) is a dNTP synthesis-inhibitor which induces double strand breaks by stalling DNA replication forks. Treating tetrahymena with HU will therefore create these double strand breaks (which we want to see if they need RSP1 to repair them). It’s handy because we can actually control and stop this induced DNA damage caused by HU by lysing the cells (which we do because we need to extra the RNA from them anyways).
The internet says that T.Thermophilia is a unicellular eukaryote. Is there a reason why you needed this specific type of eukaryote? Is it possible to use a prokaryote?
Great question, Isha! Prokaryotes do undergo DNA damage and repair, but in the larger context of our study, this DNA damage repair mechanism can have some fascinating tie-ins with cancer biology! Because this might have an effect on how we can treat human patients, working with a eukaryote will provide more relevant data that we can apply with other eukaryotes. (Also they are super easy to work, cheap, and cute! So that’s always a plus)
What process did you go through to find that RSP-1 gene expression increased by a factor of 3.71, and how reliable do you think this factor is? Would a value like this be important for understanding RSP-1 gene expression and be applicable to gauge the relative amount of DNA damage that occurred?
We used an imaging software called FIJI to quantify the brightness of our gel bands. This gives us some more accurate information as to if there really was an increase in expression for agene instead of just our eye estimation. While it can vary depending on who is doing the analysis and what specific pixels they choose, the 3.71 we got is still valid and would be similar if anyone else were to analyze our gel in the same manner!
curesymposium.org 
What information would a western blot or immunofluorescence tell us about the mechanism of RSP-1?
LikeLike
Hi there! We believe that a western blot or immunofluorescence will tell us more about the protein formation that occurs with the expression of the gene (and its mRNA) and where this activity is localized.
LikeLike
You used hydroxyurea to induce DNA damage throughout your research project. How does it work? Why this specific method?
LikeLike
Hello Maria! Hydroxyurea (HU) is a dNTP synthesis-inhibitor which induces double strand breaks by stalling DNA replication forks. Treating tetrahymena with HU will therefore create these double strand breaks (which we want to see if they need RSP1 to repair them). It’s handy because we can actually control and stop this induced DNA damage caused by HU by lysing the cells (which we do because we need to extra the RNA from them anyways).
LikeLike
The internet says that T.Thermophilia is a unicellular eukaryote. Is there a reason why you needed this specific type of eukaryote? Is it possible to use a prokaryote?
LikeLike
Great question, Isha! Prokaryotes do undergo DNA damage and repair, but in the larger context of our study, this DNA damage repair mechanism can have some fascinating tie-ins with cancer biology! Because this might have an effect on how we can treat human patients, working with a eukaryote will provide more relevant data that we can apply with other eukaryotes. (Also they are super easy to work, cheap, and cute! So that’s always a plus)
LikeLike
What process did you go through to find that RSP-1 gene expression increased by a factor of 3.71, and how reliable do you think this factor is? Would a value like this be important for understanding RSP-1 gene expression and be applicable to gauge the relative amount of DNA damage that occurred?
LikeLike
Hi Andy 🙂
We used an imaging software called FIJI to quantify the brightness of our gel bands. This gives us some more accurate information as to if there really was an increase in expression for agene instead of just our eye estimation. While it can vary depending on who is doing the analysis and what specific pixels they choose, the 3.71 we got is still valid and would be similar if anyone else were to analyze our gel in the same manner!
LikeLike