I am wanting to look at the ratio of nuclear vs mitochondrial DNA in the human cell line MCF7 cells Using qPCR (syber green and a rotogene machine using a total cell pellet DNA). I’ve designed and verified the mitochondrial DNA primers and they look great (both by a DNA agarose gel confirming amplicon size and doing an efficiency curve). For reason beyond me, the nuclear DNA primers keep failing.
I’ve ordered primers from other publications as well as designed my own. Does anyone have any ideas as to what could be going wrong? It’s not the DNA, as I just tried using different cell line DNA as got the same result. I assumed the mitochondrial DNA primers would be challenging, not the nuclear ones, so I feel like I must be doing something wrong.
any suggestions are greatly appreciate!
I should have also mentioned that I’m very proficient in qPCR for mRNA (aka cDNA) but I very rarely have looked at genomic DNA so think I may be missing something? The primers do not spam exons and since the mitochondrial primers work well I know my reagents work fine with the conditions I’m using. Such a stumper!
Hi,
normally this should not be a major challenge. You could use all primers cited e.g. for single copy loci. You might have a look here Article A quantitative PCR method for measuring absolute telomere length
Sven
Hi Cara,
since you're working with the human genome, you should test your primers in silico at the UCSC website (http://genome.ucsc.edu/cgi-bin/hgPcr). you'll see whether you choose the right sequences for primers, was wrong with the PCR protocol, or something else. it will also give you an idea on the Tm to use.
fred
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