Hello Linoy

After extraction of cfDNA, you need to design primers specific to a mtDNA gene/fragment then run a qPCR to measure and compare it within your individuals. The key point to design primers for cfDNA is the small fragment size, around 80-90 bp max!

Regards,

I guess you need to qPCR off a piece of one if the 13 mtDNA genes and get a proper control to normalize. Problem could emerge if you have a high degree of heteroplasmy.

And after I'm doing qPCR, I need to to ratio between noraml/mtDNA gene (Ct) or I need to do absolute value of copy number...what is the best way to calculte?

Also I read that it's very important to seperate the platelets from WBC and then extract the DNA...I'ts not so clear to me

Hi Linoy

In your case, since you cannot find another gene as control, and mtDNA copy number is always much more than nuclear DNA, the absolute quantification is the best approach.

some standard protocols with primers can be found here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312664/

copy number may significantly vary in pathological conditions. Regarding separation from platelets - one has to remove them if their number exeeds the number of cells in the sample several times https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063275/#pone.0163770.ref018

Hi Linoy,

You may try a NGS approach described in an article titled "Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma" ( https://www.ncbi.nlm.nih.gov/pubmed/27297799 )