The brighter band of the Rsp1 gene when DNA damage is induced indicated that Rsp1 was a lot more highly expressed after DNA damage. The faint band in undamaged DNA cell samples indicated that it was only slightly expressed. Based on our controls, we can conclude that there was more expression of Rsp1 because it is likely to play a role in the DNA damage repair pathway.
DNA is a highly susceptible molecule to chemical modifications that can lead to DNA damage. DNA damage can be induced by exogenous agents such as UV radiation, Ionization radiation, and Alkylating agents. It can also occur by endogenous agents such as oxidative DNA damage and various replication errors. DNA damage can either induced double stranded breaks or either single stranded breaks. Since DNA damage occurs from lots of different sources it is important for the cells to be able to repair this damage.
Excellent presentation, I was wondering in further directions what information will the CRISPR cas9 techniques will possibly tell you if you were to experiment further?
CRISPER cas9 can be used to change or replace a specific set of DNA or it could be used to cause a mutation at a certain location in DNA. So there can be experiments set up using CRISPER cas9 to induce mutations in proteins that are associated with sRNAs such as Rsp1. This knockout could possible validate the finding that we found in our experiment and it can be used in different types of organisms and not just tetrahymena cells. Furthermore, it can be used to correct the DNA sequences of sRNA coding proteins that could be nonfunctioning in cells which would cause the cells to be able to unable to properly repair DNA damage which could possibly lead to cancer. So being able to fix or have a deeper understanding of how DNA damage is repaired will help with future therapies for patients.
To design our primers we used online programs Primer3Plus and BLAST. The primers were then created by a company and sent back to us. We validated these primers by doing PCR and then visualizing these results using gel electrophoresis. Next we induced DNA damage by adding hydroxurea into a sample of cells. RNA extraction was then done, which was a long complicated process, but we used the manufacturer protocol for Zymo RNA miniprep. Basically, we filtered the RNA through specific filters to isolated it and added other reagents to separate the RNA. For cDNA synthesis we basically mixed a reverse transcriptase with the extracted RNA. This had a lot of steps and further reagents to help synthesize cDNA. Finally, we made a RT-PCR and visualized the results on gel electrophoresis.
curesymposium.org 
why does the rsp1 gene produce a brighter band when induced with DNA damage as opposed to the faint band in the untreated cells?
LikeLike
The brighter band of the Rsp1 gene when DNA damage is induced indicated that Rsp1 was a lot more highly expressed after DNA damage. The faint band in undamaged DNA cell samples indicated that it was only slightly expressed. Based on our controls, we can conclude that there was more expression of Rsp1 because it is likely to play a role in the DNA damage repair pathway.
LikeLike
How does DNA damage happen?
LikeLike
DNA is a highly susceptible molecule to chemical modifications that can lead to DNA damage. DNA damage can be induced by exogenous agents such as UV radiation, Ionization radiation, and Alkylating agents. It can also occur by endogenous agents such as oxidative DNA damage and various replication errors. DNA damage can either induced double stranded breaks or either single stranded breaks. Since DNA damage occurs from lots of different sources it is important for the cells to be able to repair this damage.
LikeLike
Excellent presentation, I was wondering in further directions what information will the CRISPR cas9 techniques will possibly tell you if you were to experiment further?
LikeLike
CRISPER cas9 can be used to change or replace a specific set of DNA or it could be used to cause a mutation at a certain location in DNA. So there can be experiments set up using CRISPER cas9 to induce mutations in proteins that are associated with sRNAs such as Rsp1. This knockout could possible validate the finding that we found in our experiment and it can be used in different types of organisms and not just tetrahymena cells. Furthermore, it can be used to correct the DNA sequences of sRNA coding proteins that could be nonfunctioning in cells which would cause the cells to be able to unable to properly repair DNA damage which could possibly lead to cancer. So being able to fix or have a deeper understanding of how DNA damage is repaired will help with future therapies for patients.
LikeLike
How did you perform all these experiments like removing RNA from the genes?
LikeLike
To design our primers we used online programs Primer3Plus and BLAST. The primers were then created by a company and sent back to us. We validated these primers by doing PCR and then visualizing these results using gel electrophoresis. Next we induced DNA damage by adding hydroxurea into a sample of cells. RNA extraction was then done, which was a long complicated process, but we used the manufacturer protocol for Zymo RNA miniprep. Basically, we filtered the RNA through specific filters to isolated it and added other reagents to separate the RNA. For cDNA synthesis we basically mixed a reverse transcriptase with the extracted RNA. This had a lot of steps and further reagents to help synthesize cDNA. Finally, we made a RT-PCR and visualized the results on gel electrophoresis.
LikeLike