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mitochondrial DNA copy number measurement

Qingwu Shen via methods%40net.bio.net (by shenq from purdue.edu)
Mon Sep 28 01:07:31 EST 2009


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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