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Question: You hypothesise that EV cargo from aged cells can play a role in changing the epigenetic landscape of stem cells. Outline an experimental design that allows you to explore the influence of EVs on your chosen epigenetic mark. Include hypothesis, methodology, what you will quantify, how you will interpret the data in 800 words
Hypothesis: EV cargo from aged cells can play a role in changing the epigenetic landscape of stem cells. Methodology: To explore the influence of EVs on the chosen epigenetic mark, we need to first isolate and identify the EVs. We now have several methods of isolating and identifying EVs, such as flow cytometry, nanoscale flow cytometry, single-cell sorting, and ultracentrifugation. In this experiment, we will use flow cytometry and nanoscale flow cytometry to isolate and identify the EVs from the cell culture. Once the EVs are isolated and identified, the next step is to extract DNA from the EVs. The extracted DNA can then be examined for epigenetic marks using chromatin immunoprecipitation. This can be done by isolating the DNA-associated proteins by affinity chromatography, followed by PCR analysis of the DNA. Once the epigenetic mark is identified, we can then quantify the epigenetic marks by comparing the methylation levels of the DNA from the EVs to that of the DNA from the stem cells. This can be done by using bisulfite sequencing or restriction enzyme analysis. To assess the influence of the EVs on the epigenetic mark, we can then compare the mRNA expression of the genes associated with the epigenetic mark in the EVs and the stem cells. This can be done by reverse transcriptase-PCR or microarray analysis. The data from the above steps can then be used to evaluate the influence of EVs on the epigenetic mark. This can be done by comparing the methylation levels and mRNA expression levels between the EVs and the stem cells. Interpretation of Data: The data from this experiment can be used to evaluate the influence of EVs on the epigenetic mark. If the EVs have a significant effect on the methylation levels of the genes associated with the epigenetic mark, then it can be concluded that the EVs are indeed influencing the epigenetic landscape of stem cells. Similarly, if there is a significant difference in the mRNA expression of the genes associated with the epigenetic mark between the EVs and the stem cells, then it can be concluded that the EVs are influencing the epigenetic landscape of stem cells. In conclusion, this experiment will allow us to explore the influence of EVs on the chosen epigenetic mark. The data from this experiment can then be used to evaluate the influence of EVs on the
Jan. 16, 2023, 4:42 a.m.
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