S Patnaik, Jul 2005
The fragility of RNA arises primarily from two points. Ribonuclease activity, being ubiquitous in living organisms and being exceptionally stable, can easily contaminate RNA preparations. Secondly, RNA itself is a fragile molecule. It is thermodynamically less stable than DNA because of the 2'-OH group on the ribose ring that promotes hydrophilic attack on the 5'-3' phosphodiester bond to form a 2'-3' cyclic phosphate.
The cyclic phosphate intermediate is stabilized by Mg++; thus, such divalent cations promote RNA degradation. The hydrolysis is also augmented at higher temperatures and pH.
1. Wear gloves at all the time when handling RNA or material and reagents that will be used for RNA work. Change gloves frequently and try to avoid touching surfaces that are likely contaminated (such as refrigerator door-handles).
2. Use sterile, disposable plastic ware. Look for and use 'RNAse-free'-certified material such as pipetman tips. If possible, use pipet tips with barrier (filtered tips) to reduce contamination from pipetmen.
3. Use 100% ethanol to wipe surfaces of bench, tube holders, etc., to remove contaminating microbes (and their RNAses).
4. Non-disposable plastic ware such as electrophoresis tanks and combs can be made RNAse-free by treating (soaking, spraying thoroughly, etc.) with either of these for ten minutes to an hour, and then thoroughly rinse with DEPC- or DMPC-treated water.
* 0.1 M sod. hydroxide and 1 mM EDTA
* 100% ethanol with 1% SDS (not suitable for acrylic and certain other plastics)
* commercial reagents such as RNAse Zap
* 3% hydrogen peroxide
5. Autoclaving will not remove RNAses from glassware; instead, rinse in DEPC-treated water and dry-heat at 180-200 deg for 3-4 h. Metalware can be made free by dry heating to 3-400 deg.
6. If possible, designate material such as pipetmen, electrophoresis apparatus, etc., and chemicals for 'RNA use only.'
7. Keep RNA on ice at all time, putting it back at -80 deg as soon as possible. If required, it is okay to quickly dry down samples at up to 37 deg for a few minutes.
8. Store RNA in aliquots to reduce multiple thawing and exposure to higher temperature.
9. Extract RNA as soon as possible once sample (tissue, cells) has been harvested. Otherwise, freeze sample at -70 deg or liquid nitrogen. Cells can also be homogeneized in Trizol (commercial, phenol-based RNA isolation reagent) and stored at -20 deg overnight.
10. Use RNAse inhibitors such as RNAsin in reactions that use RNA (such as reverse transcription). Such inhibitors are however expensive and also require reducing agent (usually 1 mM DTT) to work properly. Though the inhibitors are active against a variety of RNAses, they become ineffective at low or high (more than 55-60 deg C) temperature and in presence of denaturing reagents such as urea and SDS.
11. Treat solutions and water with DEPC for use with RNA. DEPC or di-ethyl-propyl carbonate reacts with amine, hydroxy and thiol groups of proteins (such as RNAses) and inactivates RNAses. Treatment involves adding DEPC to 0.1% v/v and incubating at 37 deg for half an hour to overnight followed by autoclaving. Autoclaving destroys DEPC and is an essential step. Esters may be generated during autoclaving giving rise to a 'fruity' smell (that is not coming directly from DEPC).
Note that DEPC cannot be used with chemical solutions that have amine groups, such as Tris and HEPES buffers, or mercaptans. In such cases, use DEPC-treated water to generate the solution.
Di-methyl-propyl carbonate (DMPC), a safer alternative to DEPC (known carcinogen), is used in exactly the same way..
12. RNA can be stored in 0.1 mM EDTA, TE buffer (10 mM Tris HCl, pH 7.0, 1 mM EDTA) or 1 mM sodium citrate at pH 6.2-6.6. Low pH protects against RNA degradation. EDTA chelates divalent cations that are known to chemically degrade RNA.
13. For long term storage, precipitate RNA by adding 1/10th volume sod. acetate, pH 5, and 2 volumes ethanol, and store the mixture at -80 deg. The RNA can be precipitated later by a high speed spin followed by a wash in 70% ethanol to remove the salt.
14. Certain RNA preparations, e.g., those for use as probes in in situ hybridization experiments, can be stored as 50% formamide solution to prolong their lives.