I am currently working on an experiment to analyze mitochondrial DNA copy numbers and perform bisulfite conversion using qPCR. I have a few queries and would greatly appreciate input from researchers experienced in this area:
Your guidance and resources would be invaluable for my experiment. Thank you in advance!
I can help with some of your questions.
1. qPCR (quantitative PCR) is the newer term for what used to be called rtPCR (stands for both "real-time" PCR and "reverse transcription" PCR). You can use qPCR to measure mtDNA copy numbers.
2. Exactly copy number is the most challenging assay because you need to know the exact number of template molecules in your standard curve. And you need a standard curve for both mtDNA regions of interest and a nDNA comparison. Otherwise, you can't normalize by number of nuclei/cells in the starting materials.
3. The software that comes with the qPCR machine can do the calculations. You have to set up the template so it knows which are references, unknowns, etc.
I'd suggest you start by reading the MIQE guidelines and the instructions for your qPCR machine.
Good luck!
- Badges
- Science topic
- Similar topics
- Methodology
- Research Methodology
- Research Papers