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mitochondrial DNA copy number measurement

mitochondrial DNA copy number measurement - Protocols and Methods Forum

mitochondrial DNA copy number measurement - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 09-28-2009, 06:07 AM
Qingwu Shen
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Default mitochondrial DNA copy number measurement



Hi All,

Does anybody have some experience with cell mitochondrial DNA copy number
measurement that can share with me? I need to measure mtDNA copy number of
sorted cells. So the sample cells could not be very high, with only 5,000-
10,000 cells. I need to isolate tatal DNA from cells first, and then use qPCR
to amplify mtDNA. I have following questions that need your answer or
suggestion:
1. Do you know any good kit for DNA isolation from such few cells? Also, the
kit should compatible with PCR.
2. My sample cells are isolated from mouse skeletal muscle. Do you know where
to find the mtDNA sequence for primer and probe design? I went to NCBI and just
found a couple of mouse mitochondrial genes. It's better if I can find the
whole mtDNA sequence.
3. Is there any difference in mitochondrial gene seqence between tissues?
4. This is also my first time working with qPCR. Could you please give me as
detailed information as possible about primer and probe design and ordering,
that to say, how to design and order the primers and probe.
5. Any suggestion about qPCR is more than welcome.

Thanks,

qs

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