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I precipitated bacterial RNA during 1 hour at -80ºC after depleting rRNA with Ribo-Zero kit.

Does more time lead to better results?

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  • $\begingroup$ I typically leave my mRNA in isopropanol at -70C overnight, but that's because I usually get to that step at the end of the day and I'm lazy. I would also like to know the optimal precipitation time in case I'm hurting my yield. Note that I'm not using ethanol, and different solvents may have different optimal times. $\endgroup$ – user137 Apr 7 '15 at 17:32
  • $\begingroup$ With isopropanol even keeping for 15-20 min at room temperature is sufficient. With ethanol you can keep at -20⁰C for 1-1.5-hours. Keeping at -80⁰C is not required. $\endgroup$ – WYSIWYG Apr 8 '15 at 4:00
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    $\begingroup$ You should also consider what is occurring during this precipitation step. The monovalent cations are performing two functions: (1) they are shielding the negative charges from the phosphate on the nucleic acid backbone, those negative charges would ordinarily repel each other. (2) they are decreasing the chemical potential of the water molecules in the solvent (by ordering them), so this is a dehydration effect. The alcohol also has a dehydrating effect. So if the RNA is in solution and the water is the solvent, then by dehydrating the RNA it will precipitate. When it's complete its done $\endgroup$ – mdperry Apr 9 '15 at 11:08
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As far as I know this has never been thoroughly analyzed for RNA, but there is an excellent paper on the precipitation of DNA and the usual conditions in BRL Focus by Zeugin (see below for the paper). Since DNA and RNA are pretty much the same (except for one OH-Group) and the conditions used for precipitation are also similar, I think we can use this for a very close estimate.

Interestingly neither the time nor the temperature of a precipitation have a great influence on the outcome - they change the yield only by a few percent. If you look on figure 2 from the paper mentioned, you see the following:

enter image description here

This subfigure shows that the recovery is almost not influenced by the temperature, but only by the conconcentration of the DNA (or RNA). Depending on how much RNA you expect (how much cells did you use for the isolation) I would add a co-precipitation agent when you have only very small amounts on RNA. Glycogen works very well here. Otherwise I wouldn't worry.

enter image description here

This figure shows that the incubation time has only an influence on the percentage of the recovery when then time is too short. If you incubate for an hour, you are on the safe side.

Reference:

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  • $\begingroup$ Since I usually get 100ug of mRNA from an in vitro transcription, I probably don't have to worry about time or temperature too much then? Unless isopropanol is totally different ethanol, but I doubt it. $\endgroup$ – user137 Apr 7 '15 at 23:01
  • $\begingroup$ I wouldn't worry about this. Follow your protocol and you should have plenty of yield. $\endgroup$ – Chris Apr 8 '15 at 5:33
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As many kits suggest, RNA concentration has the most profound influence on precipitation efficiency / recovery fraction. Time, temperature and precipitation agent concentration has lesser effect. See for example instruction by Life Technologies on LiCl precipitation:

The Use of LiCl Precipitation for RNA Purification

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  • $\begingroup$ N.B. Precipitation of RNA by 4 M LiCl does not include any type of alcohol (as I recall), will not work on DNA (as I recall), and is usually performed when there is still tons of rRNA present (i.e., not just mRNA) $\endgroup$ – mdperry Apr 9 '15 at 11:12

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