Preparation of RNA and DNA preservation medium

NAsafe is salting-out all proteins media of ammonium sulfate (the saturating concentration) with pH < 5 (see the lyotropic series).

The description of NAsafe (4 M (NH4)2SO4, 10 mM EDTA, 0.1 M Mes, pH 4.6):
combine 40 ml 0.5 M EDTA, 19.5 g Mes free acid, 528 g ammonium sulfate, dissolve in Milli-Q water, bring final volume 1L; stir on a hot plate stirrer on low heat until the ammonium sulfate is completely dissolved. Transfer to a screw top bottle and store either at room temperature or refrigerated.

Store NAsafe solution at room temperature. It is guaranteed for 6 months from the date of receipt, if properly stored. If any precipitation of NAsafe solution is seen, heat it to 37°C and agitate to redissolve it.

RNase inactivation is reversible; do not rinse NAsafe solution from samples before using. Blot tissues with a wipe, or pellet cells to remove excess NAsafe solution.

Guidelines for use of NAsafe solution
• Use NAsafe solution with fresh tissue only; do not freeze tissues before immersion in NAsafe solution.
• Before immersion in NAsafe solution, cut large tissue samples to ≤ 0.5 cm in any single dimension.
• Place the fresh tissue in 5–10 volumes of NAsafe solution.
• Most samples in NAsafe solution can be stored at room temp for 1 week without compromising RNA quality, or at –20°C or –80°C indefinitely.
• Do not freeze samples in NAsafe solution immediately; store at 4°C overnight (to allow the solution to thoroughly penetrate the tissue), remove supernatant, then move to –20°C or –80°C for long-term storage.

Most samples can be stored at 25°C in NAsafe solution for up to 1 week without significant loss of RNA quality. After 2 weeks at 25°C, RNA generally appears slightly degraded (marginally acceptable for Northern analysis, but still of sufficient quality for nuclease protection assays or RT-PCR analysis).

The lyotropic series salts
Some anions and cations have been noted to be effective in the order of salting-out to salting-in effect:

NH4+ > Na+ > K+ > Li+ > Rb+ > Cs+ > H+ > Mg2+ > Ca2+ > Urea > [CH6N3]+ (guanidine)

OH- > SO4-2 > HPO4-2 > F- > citrate-3 > tartrate-2 > CH3COO- > Cl- > Br- > NO-3 > SCN- > ClO4- > I-

These series known as lyotropic or Hofmeister series were related to a special physicochemical property of the ion called “lyotropy”. Ions on the left such as strongly hydrated but weakly lyotropic anions of SO4-2 are called kosmotropes, while the weakly hydrated ions, such as lyotropic anions of ClO4- are referred to chaotropes.

(NH4)2SO4 and Na2SO4 is showing the greatest effect on the salting-out (precipitation) of proteins; guanidinium thiocyanate is showing the greatest effect on the salting-in (chaotropic agent and denaturant) of proteins.

Guanidinium salts and urea are strong chaotropic salts that disrupt the structure of water and thus tend to decrease the strength of hydrophobic interactions resulting in a drastic effect on other solute molecules. For example, urea, when dissolved in water, disrupts the secondary, tertiary, and quatemary structures of proteins, and subsequently causes dissociation of proteins from RNA. Guanidinium salts and urea dissolve in water through endothermic reactions. Both guanidinium salts and urea are considered to be strongly chaotropic salts as defined by the Hofmeister series, a widely used system that ranks cations and anions according to relative chaotropic strength.

It has been believed both experimentally and theoretically that the effectiveness is more pronounced for anions than cations, which is classically used to describe the capacity of an ion to enhance or weaken the hydrogen-bond structure of water molecules, or, in other words, to enhance or reduce the solubility of a solute, respectively.

The overall salt effect depends on the nature of salts, surface, substrate, temperature and concentration. Although salt effects are very complex and quite different in different systems, lyotropic salts can be employed to shed light on the presence of molecular interactions in polymer systems.



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