Hi fellows, I've started a project in a new lab where I have to extract RNA from mice liver, brain, and muscles. Upon dissection I immediately snap-freeze the tissue in liquid nitrogen and store it for later isolation. When I had to isolate, I separated a piece of the tissue frozen in the tube with small tweezers, put it in trizol, and returned the tube to -80C.
I've been reading some places and it occured to me that the tissue must be ground to powder while it's frozen before it's used for RNA isolation in trizol. Can anyone please clarify this procedure (everything you do with the dissected tissue up until you put your sample in trizol and homogenize it)? Why is the tissue ground to powder? How long do you wait after snap freezing your tissue to grind it? What tools do you use? What do you do to make sure you preserve the RNA in your sample? How do you measure 50-100mg of tissue (Trizol protocol says thats the range u should use in 1mL) while avoiding the thawing of the tissue and activation of endogenous RNAses?
Any insight will be appreciated.
Hi Hope this helps:)
https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-guide/rna/rna-purification/rna-isolation-disruption-and-homogenization-of-starting-materials
The traditional method is to crush the tissue in a mortar & pestle, under liquid nitrogen.
Basically, get a big box of dry ice, put your mortar + pestle in that and get both really cold. Get your tissue out too, keep that on dry ice.
Label some tubes appropriately to hold the powdered tissue, chill those as well.
Also get a metal spatula that fits into your tubes, and chill that.
TL:DR version: DRY ICE CHILL ALL THE THINGS
Now carefully tip your tissue into the cold mortar and tip some liquid nitrogen on top of that, then (carefully!) grind up the tissue to a fine powder -I usually tap it a few times to break it up into smaller chunks (most frozen tissue is very brittle) and then grind those. Add more liquid N2 as necessary (carefully: no surface tension, so it slops around easily). Once your tissue is fully powdered, let the liquid N2 evaporate (won't take long) and scoop the powder carefully into the pre-labelled, pre-chilled tubes.
Then freeze these.
The method I use now is this bad boy: https://cellcrusher.com/
Which is the same principle, but substitutes careful mortar and pestle work with just putting the tissue in a dry-ice chilled metal crusher and smashing it with a hammer (also quite therapeutic).
For RNA isolation, I take a second set of tubes, pre-chill those on dry ice too. I then scoop (chilled spatula) some tissue into those -one or two decent scoops is ~100mg tissue- and add 1ml of trizol directly to the frozen powder. Seal the tube, vortex well and then keep at room temp for RNA isolation.
The idea is that your tissue, once frozen, never, ever defrosts until you directly add the trizol.
Hello, You don't necessarily need to pulverize your samples before extraction with Trizol; I have seen this procedure being used more with plants. For animal tissue, you usually add Trizol directly onto the tissue while it's still frozen, or just thawed (preferably still frozen).
In our laboratory, we usually aliquot enough tissue mass for extraction (between 50 and 100 mg) into a new tube free of DNAses and RNAses, and immediately add 1 mL of Trizol. This sample is then homogenized using the BioSpec Tissue Tearor (we've tried other brands and never got good results) or a pestle. To ensure that you preserve RNA, it's important to follow the protocol steps rigorously, minimizing any contamination by endonucleases as much as possible. Use gloves and reagents suitable for molecular biology and good laboratory practices. With mouse tissue, you probably won't have any problems. The link recommended by Uma Dharshini K Vijayamuthuramalingam is very worth reading. You should also read the complete Trizol protocol, Troubleshooting, and FAQs.
Sincerely,
Eli Farber
Hello,
Once you dissected the tissue, just grind it with the pestle and mortar by adding the liquid nitrogen to it and kept at -80. Whenever, you start the RNA isolation experiment take 30mg of grounded tissue and process with Trizol or Isolation kit in aseptic condition with proper precautions.
You'll get better yield.
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