I am trying to compare mtDNA levels in a mutant strain relative to N2, and I don’t need to know the absolute mtDNA copy numbers. All the protocols I read lyse one worm per tube, and use a standard curve to find the mtDNA copy number of that single worm.
Instead, I would like to know if it is possible to wash off same-staged worms, extract the DNA and use a mtDNA-specific primer set to simply compare mtDNA (normalized with nuclear DNA) levels in mutant vs. N2 worms?
In other words, is there any other method to compare mtDNA levels between strains with a bunch of worms that does not require creating a standard curve first?
That’s not going to be possible with the resources I have. Could the Trizol method be modified to separate DNA from worms, and use the Applied Biosystems 7500 qRT-PCR machine to quantify mtDNA?
Thanks for the information as always, Steve. I wanted to see if it can be done the way I do RT-PCR starting from RNA isolation using the Trizol method. I’ve never had to isolate DNA, so I’ve just begun looking into the protocols.
Southern blot is definitely an option, a very good option; I just was curious to see if any lab has quantified mtDNA using qRT-PCR machine but without first creating a standard curve.