Hi, I'm a new master's student who has been studying mouse IPF model, and I read that Trizol with tissue needs to be cold.
But I can't keep the tissue cold while I homogenizing it with Tissuelyser ll, Could you share me a tip for extracting RNA with the machine?
Hello Eunseo Yeo,
In such case, I feel you should cool the sample tube on dry ice or in a freezer to approximately -80 degree C which may be sufficient to prevent the tissue from thawing during the disruption process.
Best.
It doesn't need to be cold.
The point is that tissue lysis results in liberation/dysregulation of many, many cellular RNAses that will promptly start chewing away your RNA.
This cannot happen if the tissue is well frozen (which is why frozen tissue at -80 is good for RNA isolation even years later), and also cannot happen in trizol (since the whole point of trizol is to unfold/denature/destroy anything that isn't RNA).
The trick is to go from "frozen tissue, not in trizol" to "homogenised non-frozen tissue in trizol" as quick as possible.
In your case, the tissuelyser will do _most_ of the work very quickly, so really it comes down to how big (and how tough) your tissue is.
If you have small pieces of tissue that are fairly soft (liver, lung, etc), then these are good to go as is. If you have massive chunks of tissue (or something tough like skin), I would consider crushing them a bit first (mortar and pestle, chilled in liquid nitrogen/dry ice).
Then when you're ready to roll, just add room temp trizol + lysis beads straight to the frozen chunks and quickly move to the giant angry smashy tissuelyser machine. As long as you're quick (
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