I'm planning to perform a DNAse hypersensitivity assay followed by qPCR. What kind of control primers do I use to normalize my data? When I do regular qPCR to study gene expression I use GAPDH primers.
Can you describe your project a little more? If you are "mapping" a particular area, each primer set you use should be compared to the uncut DNA sample. You would then compare DNAse sensitivity ratios for each cellular condition you are testing. Make sure that your "undigested" sample gives a good qPCR signal. I have found, as well as others, that qPCR on genomic DNA is sometimes inhibited. A quick sonication (10 seconds) can break up the DNA randomly enough to increase your uncut PCR to its "maximal signal".
I don't understand what do you mean. I think DNAse hypersensitivity assay are completely different from qPCR. In gene dosage assay you have to use genes like PMP22 that 2 normal alleles of it are necessary in development. some genes like GAPDH are only good in gene expression analysis.
Gary: Thanks for the answer. I want to transfect cells with a plasmid encoding the protein Im working with, then check a specific genomic area that Im intersted in using DNAse HS assay coupled with qPCR. So basically I should normalize against the uncut DNA if I understood you right.
Feyzollah: Im not performing gene dosage assay. You can couple DNAse Hypersensitivity assay with qPCR to check the results of the Dnase treatment.
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