Nucleic acid preservation

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    • #9341
      MrMistery
      Participant

      I have been told at the lab that you can store DNA at -20 Celsius with no problems, but you need to store RNA at -80 Celsius. Why is that?

    • #83023
      biohazard
      Participant

      I think this is mostly due to the much better structural stability of DNA when compared to RNA. After all, they can isolate DNA from frozen sources that are thousands of years old. What the exact mechanisms of RNA degradation in frozen samples are, I’m not sure. At least in mRNA, such structures as the poly-A tail seem to be very labile and degrade spontaneously, maybe this applies to some bonds in the RNA as well (U vs. T?).

      Also, RNAse enzymes, which are very abundant inside cells as well as in the environment, rapidly degrade RNA. Maybe they retain some degree of activity even in frozen samples if they are located around (i.e. next to) the RNA strands, and this can be completely stopped only at -80C or colder. I think that at least in food spoiling some enzymes remain active even in frozen food, so apparently certain enzymes can function in frozen environments (I once enquired a professor of food chemistry about this).

    • #83028
      blcr11
      Participant

      For RNA, the adjacent hydroxyls on C2’ and C3’ of the ribose are susceptible to both acid and base catalyzed beta-eliminations. When the 2’ hydroxyl is absent, as it is in DNA, the elimination reaction is much less fascile. Water is a strong enough base to catalyze the elimination from RNA—albeit slowly. You store RNA at the low temperature to reduce the spontaneous eliminations you would get at the higher temperatures. RNases are also ubiquitous, but if you haven’t taken pains to keep RNases out of your sample from the beginning, your RNA will be trash anyway by the time you are ready to store it. DNA is stable toward base-catalyzed elimination, which is why you do alkaline lysis to purify DNA plasmids; DNA is stable under those conditions while RNA is not.

    • #83029
      Cat
      Participant

      I would like to add that your lab is just being extra cautious; our lab keeps it at -20 just fine. If you get RNase into the sample during prep, most RNA will be degraded before sample gets frozen. Storage at -80 is recommended though for all RNA and DNA samples kept long term.

    • #83037
      MrMistery
      Participant

      Thanks for the answers guys.
      I didn’t have any RNase present in any of the samples, although 4 out of 18 turned out contaminated with DNA. We were extra careful to get rid of all the proteins in the sample, and always keep the tubes on ice(I heard dry ice is much better, but we don’t have that). Also the storing was only for 12 hours, we did reverse transcription the next day. What biohazard said about the poly-A tail might be especially important, as we did reverse transcription of mRNA using poly-T primers.

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