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If I extract RNA from a (leaf tissue) sample using a one-step phenol:chloroform extraction, how long can those samples be stored at -80°C? And how many times can I defrost and refreeze them before they will become degraded?

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I'd guess that this will depend heavily on the purification of the sample, pure RNA (no Mg2+ e.g) can be stored for a very long time at lower temperatures. Pure RNA in water can be stable for months at 4°C. Freezing and thawing RNA can affect the folding (for RNA with important tertiary structure) as I have observed. –  Mad Scientist Feb 10 '12 at 14:12
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If you're worried about freeze/thaw cycling, aliquot them into multiple tubes -- that way you only thaw what you need to use. –  jp89 Feb 10 '12 at 19:49

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I've found that extracted RNA using commercial kits has stayed stable for many years at -80 C. I would certainly aliquot it before freezing however as RNA is particularly sensitive to freeze-thaw cleavage.

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We've actually checked the degradation of viral RNA extracts, and they will for the most part hold up for more than a year at -80. We did see more stability variation correlating to sequence than we expected, however. –  Atl LED Jul 13 '13 at 3:23
    
This is interesting - how did you measure the degradation? I imagine degradation that might have a significant effect on, for example, RNASeq, would not show on a gel. –  Richard Smith Oct 29 '13 at 16:40

We can keep extracted RNA in -80°C for a few weeks, but before the start of any experiments, it needs to be validated by gel electrophoresis.

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I keep my RNA in 1mM sodium citrate pH 6.4. Citrate is a chelator, and helps trap the divalent metals many RNAses need to work. The lower pH also helps inhibit RNAse activity. EDTA could work as a chelator, but it only chelates well at pH's where RNAse activity is worse. Citrate gives both chelation and low pH.

Of course I also keep my RNA at -80.

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Do you need to do anything special to remove the citrate when it comes time to use the RNA, for example to make cDNA? –  Richard Smith Oct 29 '13 at 16:39
    
I've never noticed any problems, but I've never really checked. Only been working with RNA for a couple months now, and my lab is in an old building with a lot of dust, so RNAse contamination has been a bigger issue than enzyme activity. An isopropanol precipitation should be enough to clean up the RNA before any reactions if citrate proves to be a problem. –  user137 Nov 1 '13 at 19:58
    
We're also in an old dusty building, and RNase is a problem for us too. Thanks for the tip - I'll check whether it works for RNAseq purposes. –  Richard Smith Nov 4 '13 at 22:26

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