Tissues in question :surgically removed granulomas and cysts from persistent apical periodontitis. Tissue has varying degree of fibrous content. Current train of thought is cryogrinding in liquid nitrogen chilled metal tube, then add Trizol to my crushed samples and proceed as protocol dictates. Maybe I should also use a sonicator when the tissue is dispersed in Trizol to further lyse the tissue? Thoughts?
Hi,
In our lab we've been the gentlemacs dissociator and Trizol for gingival cells (Human and murine) with very good results. If you don't have that equipment around, I think that the cryogrinding is your best option. But try to work with small volumes and with the smallest grinder that you can get.
i do not have any experience with sonicator.
Good look!
We have been very successful with the SPEX 6775 Freezer/Mill® Cryogenic Grinder with the micro tubes for tissues containing high amounts of cartilage. Results in a fine powder. Trizol can then be added directly. Not need for sonication.
Thank you both. I cannot justify the expense associated with acquiring those machines for my simple project, but I found out by experimenting that manual cryopulverizing followed by ultrasound sonication in Trizol that this method worked well for protein isolation. I must, of course, verify my findings by running my Luminex xMAP analysis, but I could visually inspect the precense of protein-pellets after all washing steps per protocol, so there is hope. Trudy: Is there truly no gain in sonication? I mean, cells in suspension could be further lysed by this, and in my very small sample sizes I would go for maximum gain. Thoughts?
Trizol is so effective that any additional steps only result in loss. Trudy.
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