Can DNA be solubilized in hydrochloric acid? - (May/12/2009 )
I have in vitro cultures containing mineralized bone nodules from differentiated rat bone marrow cells. I am measuring the amount of calcium deposited in the matrix by solubilizing the calcium in 0.6 N hydrochloric acid overnight at 4oC.
What I would like to do is to normalize the amount of calcium to the amount of DNA present. Would the HCl be able to solubilize the DNA from the cells, and hence I can measure the DNA in the supernatant using some sort of fluorescent nucleic acid dye (e.g. DAPI)?
Or if someone has any other protocol to measure matrix calcium and DNA in the same sample it'd be greatly appreciated!
HCl, especially at that molarity, will quickly depurinate the DNA. But if you are simply looking for amounts, it might be possible to quantitate it. The major problem will be separating it from everything else. Neutralization, followed by a phenol/chloroform extraction and ethanol precipitation would be the place to start. I'd be nervous about how quantitative these approaches would be, however. Do you have a source of good "identical" controls that you could use to develop a consistent process? A perhaps easier approach might be to neutralize, followed by a pico-green fluorescent assay.
The HCl should depurinate the DNA via acid hydrolysis, which will mean that you will have fragments. Small sections of DNA can be very hard to extract due to the size not being easily precipitable and will not make for easy detection with any DNA intercalating dye, such as DAPI, Ethidum bromide, sybr-green etc.