I'm looking for the best performance to extract RNA from some tissues which are stored for more than 1 year or even a couple of years in - 80 degrees of Celsius and we are not sure about its safety in storage.
Given the time of storage, you might expect to have some loses, so provided you have at least 50mg of fresh tissue I would suggest a quick and easy Trizol extraction kit protocol.
They are easy, fast and efficient with the ability to extract even small RNAs. Make sure you aliquot your samples and use liquid nitrogen to keep them cool at least until the Trizol step. Here's a "sample protocol". Remember each company has minor differences on their protocols, so make sure you follow directions exactly.
http://tools.lifetechnologies.com/content/sfs/manuals/trizol_reagent.pdf
Best of luck!
Hi Aref,
I regularly extract total RNA from samples that have been in cryostorage for a long period of time - in some cases decades. Granted, cryostorage is different to -80. I grind my tissue up in a pestle and mortar under liquid nitrogen - i.e. I pour LN into my pestle and mortar (dangerous), then add my tissue, then grind it until I get a pinkish powder. Note that some tissues take quite a bit of a effort to break down! Then, when the liquid nitrogen has almost boiled off, I decant this Liquid-nitrogen/Tissue mix into a 50ml falcon tube (pre-cooled in LN to reduce chance of cracking; also dangerous). Once the remaining LN has finally boiled off, I add a couple of mls of Trizol, then continue the Trizol plus protocol as normal. This approach minimizes any additional RNA degradation, and has become the standard protocol I use.
I also once explored the possibility of using Invitrogen's RNALater ICE, which is a product that supposedly allows you to defrost your ancient samples without degradation, but I found success to be dependent upon which specific tissue you used, so I did not continue with this.
Trizol plus link:
http://www.lifetechnologies.com/order/catalog/product/12183555
RNALater ICE link:
https://www.lifetechnologies.com/order/catalog/product/AM7030
Hope that helps,
Gareth
thank you all ...
but for some reasons I prefer to use RNA x plus to extract...
Hi Aref
I extracted RNA from plant tissues which are stored for more than 7 months in - 80 degrees by using RNX-plus and I got good results. CinnaGen company protocol was used.
hi mrs.matlobi
thank you for your reply ... I'm using old tissues which are stored for more than 2 years in -80 degrees of Celsius and every time I had tried to extract the RNA I had a problem ... and even some times I lost my RNAs totally ...
Isopropanol must be cold. was it in -20 degree for 12 hours at least?
It is very important!
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