Hi all,
this is another of my mysterious questions. I am working with brain chromatin during development, in particular with P4 and P60 mouse brains, and I am always getting a weird result. whether I am preparing chromatin for ChIP or just isolating it after Mnase digestion, I always get a huge amount of DNA from P4 brains but very small amount from P30. Before you ask I treat every sample with almost 200ug of PK for 2 hours. My first thought was that DNA could get stuck to proteins and so I could lose it during my phenol chloroform extraction. I always start from same amount of nuclei so DNA amount should be same. Anybody can help me solve this?
Dear Gilda,
The key players are polyamines (PAs), spermidine and spermine. Why? PAs work as casks to hold DNA. This stabilizes DNA structure and an extraction of DNA from brain is a process which should count (i) PA unbinding from DNA procedures and (ii) cell types you are isolating: (1) neurons versus (2) glial cells. Since in adult brain Glia outnumber neurons about 13 times in brainstem and ~3.5 time in cortex, so the nuclei of astrocytes, NG2 cells, oligodendrocytes, ependyma (all glial cells) contain most of DNA sampling from brain. Are you sure that the samples of collected nuclei are containing both cell types or you are loosing one of the types due to just isolation procedure?
Another key is the age. While young brain is not much differentiated (glia versus neurons), after establishing adulthood (for rats and mice it is from 21 day to 60 days) the glial cella are maturated and glia outnumber neurons. Therefore, how do you collect nuclei and separate them, glial versus neuronal?
Finally, In the cells ~80% of spermine and spermidine are bound on RNA and DNA. During development (and specifically during aging) PA amount is dropped down (the well known fact). As so, the amount of chromatin isolated will be also dropped down. For example your problem may be related to the concentration of PAs in your samples and therefore to amount of DNA isolated. Check your methods, how does it related to PAs cleavage from DNA, etc. Does my answer helps conceptually?
Cordially, Serguei.
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India