Brain tissue that has been fixed during perfusion with 4% PFA and stored for several months in 30% sucrose, 1X DBPS solvent for both solutions.
Wondering whether it's possible to extract DNA from this tissue, any special methods of doing so? To date have only extracted from fresh tissue using the DirectAMP tissue extraction kit by Denville.
I've seen some posts about extracting from tissue stored in PFA/formalin, so would appreciate any insight into whether the same methods for DNA extraction could be used here or not due to storage in sucrose.
Thanks!
A homogenizer machine, beads, or a mortar and pestle may grind your sample down well enough. The type of homogenizer depends on the size of your samples. Try soaking samples, cutting the samples into smaller pieces, and grinding it down with homogenization.
Thanks, Tatiana. However the size of the tissue is not my primary concern. I was wondering specifically about the chemical changes to DNA structure caused by fixation in formaldehyde and how those might be remedied by an adapted DNA extraction protocol.
Unless another suggestion comes in, I'll be trying my hand at the procedure outlined here: Article DNA Extraction from Formalin-Fixed Material
Looks this protocol might work since they have published it. I will avoid autoclaving, rather heating at 100C for 40 min or less, I know in PCR, we do heat DNA upto 95C confortably and that works for amplification.
The protocols that may help elongate DNA fragments are through heating, a deparaffinization protocol, and then applying the DNA extraction kit. AFA or adaptive focused acoustics could help as well.
The cross-links between DNA and protein due to the fixation seem to be a hindrance for extraction. The chemical changes to DNA fixed with formaldehyde lead to DNA degradation. The aldehydes cause DNA to degrade at 4 degrees celcius. So, extracting DNA may require buffered formalin or alkali treament. The heating and buffered formalin reverses those DNA to protein cross- links.
Immerse tissues in buffered formalin for 1 day.
Heat tissues for 30 minutes at 95 degrees Celsius.
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