This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes again from the “RNA Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but they do not represent endorsements by, or the opinions of, BioTechniques.
Can I safely store purified RNA at −20°C? (Thread 14901)Q I normally store purified RNA at −80°C in DEPC (diethylpyrocarbonate) treated water, as many protocols suggest. But I would like to be able to store purified RNA in a −20°C freezer. Is that a problem? What solution is suitable for storing RNA at −20°C?
A1 You can store RNA as ethanol precipitate at −20°C. But before you can use it, you have to spin it down, rinse with 75% ethanol, air-dry and redissolve it in water.
A2 That's a good method, but be sure not to have Mg or Ca ions in the preparation. Freeze-thaw cycles with these ions lead to strand breaks.
Q Does RNA degrade when stored at −20°C in water or TE [tris(hydroxymethyl) aminomethane-ethylenediamine tetraacetic acid] solution?
A Depending on the type of RNA sample, you can store it at −20°C in water or TE without significant degradation for a long time. For example, in vitro transcriptions of short RNAs are quite stable, but total mRNAs extracted from cells or tissue can be more susceptible to degradation.
For long term storage, ethanol precipitate will definitely be a more reliable choice.
Q Does it work with isopropanol, too?
A It should be OK. But you'll have to rinse the pellet with 75% ethanol. Ethanol is more volatile, and the pellet can be air-dried more easily.
Q If freeze-thaw cycles affect RNA degradation, wouldn't storage at −20°C be okay, as long as the sample remains frozen without thawing?
I wonder what degrades RNA molecules even at −20°C.
A1 Some enzymes such as proteases are still active at −20°C. I am sure RNase can slowly degrade RNA at −20°C. That's why storage at −80°C is recommended.
A2 I've kept RNA at −80°C in water and then successfully used it for RT PCR years later. (I didn't need to quantitatively run it against more recent RNA, but for my purposes it was fine.)
A3 Stability of RNA at −20°C depends on the purity of the sample. Pure RNA samples have been kept at −20°C in our lab for years and still give good RT results.
A4 I usually store my RNA samples in RNAse-free water at −20°C, and nothing untoward has happened in the past 3 years.
I did once have to send RNA samples to South Korea, though, and twice it got degraded before it reached there. To figure out what had happened, I tried keeping RNA in ethanol at 37°C for a week. The sample looked fine on the gel, but when I added NaAc and stored it again at 37°C, it degraded. So I think the storage solution matters more than the temperature.
A5 Samples can last at −20°C for several years, as long as you don't thaw them. Because −20°C freezers are opened so frequently, and because some models are frost-free (which means they increase temperature automatically on occasion), they are not really safe for saving your samples.
