I extract RNA using TRI Reagent (Phenol-Chloroform based method), the RNA pellet was washed by adding 75% ethanol and dry the RNA pellet for 5–10 minutes by air-drying. Then, the RNA was dissolved in either RNase free water or ultraPure DEPC-treated water and stored at -80 C. I used to test the quality of the RNA sample that stored at -80 C more than 2 years by gel electrophoresis and full-length cDNA amplification, it still intact.  

Thanks Akechai, that is indeed what many do, but it is (more or less) general consensus that for long term storage it is better not to use H20 alone. I think the problem is that when you thaw your sample, if everything was not carried out completely RNase free, you have no protection whatsoever against degradation 

Thanks David, your explanation is very interesting, although I am not sure I am knowledgeble enough to fully understand it. I would like to ask you:

- what would be the consequence of 75% ethanol vs 50% regarding the enrichment in water that you mention? Maybe that only with 50% you get to evaporate enough ethanol without causing excessive dehidratation of the pellet?

- I actually did not assume that with higher concentrations of EtOH I could mantein the RNA in solution, but if it precipitates (and I believe it will)  what is the problem? It is not what we want when we add Na aceteate afterall? My purpose was trying to avoid to add Na acetate and the annoying washing to remove it after that. Could not I bring the RNA back in solution with higher temperature and mixing well?

- Would be viable using the typical salt-free 50% ethanol-water if a vacuum dryer is not available, or is it a bad idea?

Thanks a lot 

Hello Paolo,

I think it depends on what you are after, industry standard, core service standard or keeping the RNA for a project. Note anyway that if you do not work for sure RNAse-free, it will not matter how you store the tubes, because the RNA will get degraded after, when you use it. Perhaps you could store your RNA in the presence of an RNAse inhibitor, to protect it afterwards. Ribonucleoside Vanadyl Complex are fairly cheap, for instance.

I wonder if the main reason for storing in ethanol is to minimise the effect of freeze-thaw cycles.

I have worked in projects where we kept our RNA samples for 4 years in the -80 in water, and they were just fine. What I would do, as David suggests, is aliquoting to avoid freeze-thaw cycles, if you plan on accessing those samples often. About RNAse, unless you have a good reason to wary (e.g. someone blew open a bottle of RNAse powder in the lab), I would say that working in a rationally clean environment with clean tips, gloves, tubes and reagents will prevent 99% of the cases of RNAse contamination. It's hard to believe, but I have seen gut-wrenching scenes of people leaving their tip boxes open by open windows, working with plants with soil and fungus and parasites all over on the bench (oh my), and their RNA was still fine (but don't ask me about their results, I would not trust them!).

Best,

Ruben

Ruben, unfortunately aliquoting would be a huge problem, because of lack of storage space

David, your answer is great and made me really think !  But now I have another doubt ! If I have a stock of RNA in 50% EtOH, and I withdraw a larger aliquote, my only chance would be to wait for the EtOH to evaporate, or can I pellet it somehow?? I doubt that even spinning it hard, would be possible to precipitate from a 50% solution. What could / should I do in this case? Would be enough adding a number of Volumes of 100% EtOH + hard spinning + chilling? I suspect that the only way to pellet the RNA and pipetting out the EtOH would be by the classic EtOH + Na Acetate precipitation. If this is the case, and to avoid the use of salts and the relative washing steps, best option for me would be indeed having very concentrated stocks (like you were maybe suggesting), so that the aliquotes I could possibly need would be in the order of 1 to few microliters, and removing the EtOH would be quite easy even by air-drying 

Hello Paolo,

If lack of storage space becomes a problem, you could consider making aliquots in PCR tubes or plates. I would think that very small aliquotes (few ul) might not be ideal, because they would evaporate or sublimate more easily (if RNA concentration is important here).

Also, if storage space is a problem you can change your approach altogether and store at room temperature. Check the Gentegra products:

http://www.gentegra.com/products.html

And Illumina's assessment (I think Gentegra is part of Genvault):

https://support.illumina.com/content/dam/illumina-marketing/documents/products/whitepapers/whitepaper_genvault_genotyping_recovered_dna.pdf

And the patent http://www.google.ch/patents/US20150267245

Best,

Ruben

Thanks Ruben. The fact is that making aliquots become cumbersome, both in terms of spaces than in term of managing many samples. PCR tubes would require small aliquotes anyway, and I doubt they would seal very well, allowing evaporation.

Gentegra that you suggested looks very interesting, and I will consider in the future, but I do not think it is an option right now, even in term of extra costs.

My yield in term of isolation is not fantastic, but still a minimum of 80 ug of RNA for each Trizol reaction, and I have no need of big amounts of RNA for downstream retrotrascription, let's say 0.5 to 1 ug are plenty. Doing some math, if I do not miss anything, I can get 80 ul stock Volume (50% Water Ethanol) with a concentration of at least 1 ug / ul.  If I understand properly David's explanation, I could just withdraw a very small amount of stock, let's say few microliters, and easily dry them (or maybe it is not even necessary to bother) and having enough for gel electroforesis and cDNA syntesis 

Hello,

Perhaps you could use methanol instead of ethanol, since RNA will be soluble at higher concentrations in the former. Since you don't really want to precipitate, methanol will air-dry faster than ethanol (if you don't have a vacuum drier or vacuum-centrifuge). In that case you could draw a small aliquot from a concentrated RNA methanolic stock, air-dry it and perform the RT directly in the tube right after. Anway, >1ug/ul stock is superb, you have more than plenty RNA for your application, wow.

Beware of heating the RNA at >60 degrees, because in the presence of water you will have hydrolysis, which will be worse if you have divalent cations around. Unless some tests show that RNA is OK, air-drying would be safer.

Best and good luck!,

Ruben

Thank you Ruben, yes, I also  thought that maybe was a good idea to work directly in the tube without trying to re-suspend that 1 or 2 ug of RNA I need for the RT or for the gel. Thanks a lot about the methanol, seems very interesting option! I will try for sure the air-drying and will try to avoid any risky heating. I am not still sure what would be the better tubes to store my sample at -80, I need something that seal tightly, as samples needs to stay there for years, and possibly a small screw cap tube suitable for 50-80 ul of samples, it seems that most tubes are meant for 1 or 2 ml !!