Hello everyone,

I'm currently working on calculating the relative levels of mtDNA using qPCR by comparing the Ct values of a mitochondrial gene (nd1) and a nuclear gene. According to my understanding, the Ct value for the mitochondrial gene should be lower than that of the nuclear gene. However, I am observing the opposite in my experiments, with a Ct of approximately 28 for the mitochondrial gene and 21 for the nuclear gene.

My specific questions are:

For context, my DNA extraction process involves treating the sample with proteinase K for 30 minutes, followed by centrifugation and overnight precipitation with isopropanol at -20°C. I then centrifuge again and wash the pellet with ethanol. The primer sequences I am using have been previously validated by other research groups.

Thank you in advance for your insights and suggestions!