Hello, hoping someone can help. I've been snap freezing very small (~Day 10) organoids to do RNA extractions later, since I don't always have time to do it at the time of harvesting. I didn't get on very well using RNA later so have just been snap freezing them on dry ice.
What's the best way to thaw them to maintain RNA integrity? Has anyone tried RNA-later ICE?
I'm planning to use an RNA micro plus kit and do bulk RNAseq on pooled samples.
Thanks in advance!
How small is "very small"?
It may be quickest (and easiest) to simply add your lysis buffer (RLT) directly to your frozen organoid and then immediately smoosh it with a plastic micropestle or similar.
Don't thaw them at all beforehand: literally thaw them directly in the lysis buffer (this immediately inactivates RNAses).
You dont want them to slowly melt in GiTC. If they are small enough as John suggests is probably best. If they are bigger, buy a small high speed tissue homogenizer to speed it up. Sorry about the RNAlater, that was a long time ago that I did that. Don't bother with RNAlater ice.
John Hildyard thanks for getting back to me. If you imagine 10-20ul in an eppendorf that's about how small 10 organoids at this stage is.
I'll do what you suggest, sounds like I can get away with adding the lysis buffer straight away them and homogenizing them with a syringe since they're so small. Hopefully the snap freezing was ok.
Thank you!
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