Hi all!
I would like to determine if there is leakage of mitochondrial DNA to the cytosol in cells (after oxidative stress) by qPCR by doing nDNA/mtDNA. According to the protocol in the reference, I have extracted cytosolic mtDNA and nDNA from cells.
But, the results of my experiment were not as expected.
Do I need to ensure that the concentration of DNA in each well is consistent？ If the nDNA of each sample is diluted to the same concentration, such as 1 μg/μl, should my mtDNA change by the corresponding multiple with nDNA?
Thank you.
Unless there is a protocol I don't know about where you can get just the "leaked" mtDNA and nDNA, but not the mtDNA from inside the mitochondria, I don't think your experiment is plausible.
One, it's Dr. Burnette.
Two, that article only shows a potential "indirect measure of cytosolic mtDNA" using protein markers, not a mtDNA isolation protocol.
Three, go talk with your advisor. Your project is not feasible by qPCR.
This is your research project. You are responsible for knowing the literature in detail. You have an advisor, go talk with them.
You should read more about qPCR. Start with the MIQE standards to learn what is needed for reliable, publishable data.
If you have a specific question about qPCR, then come back and ask again.
wow she is so rude. im just amazed on the responses. you could try to read this paper "Assessing mitochondrial DNA release into the cytosol and the subsequent activation of innate immune-related pathways in mammalian cells"
i hope it could help you.
this platform is to help each other as a researcher to overcome any difficulties. not to flaunt your degree.
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