I had stored tissue in RNA later at -80 degree for RNA isolation. Now, i wish to culture cells from that very tissue sample. Is that possible? If so then how?
I really doubt it.
It's a mystery mix of chemicals designed pretty much solely to permeabilise tissues and cells and gum everything up to prevent RNA degradation. And then you've also stored this tissue at -80 with no additional cryoprotection.
My guess would be "your cells are all dead within minutes of adding RNAlater, and if not, they are certainly all dead after being frozen at -80."
Culturing primary cells from tissue is tricky enough even with freshly isolated tissue. Attempting to do so with defrosted tissue floating in RNAlater seems like a waste of time.
I really doubt it.
It's a mystery mix of chemicals designed pretty much solely to permeabilise tissues and cells and gum everything up to prevent RNA degradation. And then you've also stored this tissue at -80 with no additional cryoprotection.
My guess would be "your cells are all dead within minutes of adding RNAlater, and if not, they are certainly all dead after being frozen at -80."
Culturing primary cells from tissue is tricky enough even with freshly isolated tissue. Attempting to do so with defrosted tissue floating in RNAlater seems like a waste of time.
Hmmm its best to use fresh tissue and freshly isolated RNA from tissues since its a problematic process. I dont think you would need more cryopreservation as long as you have followed RNA later procedure.
BUT
"4) Which downstream applications can be used with tissue stored in RNAlater?
Tissue stored in RNAlater is compatible with all commonly used RNA isolation methods, including single reagent isolation products like Trizol®, and all of Ambion's RNA isolation kits. It is also possible to extract both genomic DNA and total protein from samples stored in RNAlater. RNAlater will denature proteins, so it is only compatible with routine protein analyses such as Western blotting and 2D gel electrophoresis that do not require native protein."
Check this out please:
https://www.thermofisher.com/tr/en/home/references/ambion-tech-support/rna-isolation/tech-notes/rnalater-faqs.html
"1) How do I use RNAlater to store my tissue/cell sample?
For fresh tissue, simply cut samples to a maximum thickness of 0.5 cm in any one dimension and submerge in 5 volumes of RNAlater.
Cultured cells should be pelleted, resuspended in a small volume of PBS, then mixed with 5-10 volumes of RNAlater.
Once in RNAlater, samples can be stored for up to 1 day at 37°C, 1 week at 25°C, 1 month or more at 4°C, and long-term at -20°C or -80°."
How long has it been since you stored? If it is less than a month I would try my chance. Also if the tissue is big there is a chance it may have good cells stored in the core part of the tissue.
I agree with John that it is very unlikely that the cells in your sample are still alive!
Thank you John, EC and Theodora for the reply. I had kept the samples at -80 degree around 6 months back so i think it will not be possible to get any live cells.
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