Basically, RNA can be stored in purified form after its extraction or synthesis, or unprocessed samples containing the RNA of interest are stored. However, even in the latter case, proper care must be taken to avoid (re) contamination of RNA with RNases once it is extracted from the stored sample.
Little information is available on the long-term stability of isolated and purified RNA during storage. Most studies have been carried out focusing on various storage conditions of unprocessed samples before RNA extraction, or the material was stored at the stage of lysed cells or tissue but before RNA was isolated.
Depending on the extraction method used, purified RNA is either being eluted directly from a filter or column after ion exchange chromatography, or it is obtained as a pellet after precipitation with ethanol or isopropanol. Irrespective of the storage conditions, it is of great importance that all materials (glassware and plasticware, chemicals, water, etc.) are free from RNase contamination.
The most common way to store RNA is by dissolving it in RNase-free water and freezing at — 70°C. This bears the advantage that the RNA can be used virtually for any subsequent experiments without having to worry about interference of the storage medium with the reaction conditions in the experiments. However, RNA stored in water is prone to degradation because of unfavorable pH, as well as traces of RNases. For this reason, RNA should be dissolved in or eluted with a low salt buffer such as 1 mM sodium citrate or TE (10 mM Tris, 1 mM EDTA) at pH < 7, thereby preventing base hydrolysis by chelating free cations present in the RNA preparation.
If the higher-order structural integrity of the RNA is crucial, it should be stored in a buffer with an increased salt concentration to maintain hybridization of double-stranded RNA as well as self-complementary sequence elements (stems, pseudoknots) in single-stranded RNA. This is particularly important if such RNA is to be analyzed for secondary structure elements by targeted chemical/enzymatic degradation.1-4,5-1
Stabilized formamide (FORMAzol®, Molecular Research Center, Inc., Cincinnati, OH) can be used to dissolve precipitated RNA. It allows to store the RNA for at least 2 years at — 20°C, whereas RNA dissolved in aqueous solutions should always be kept at — 70°C. However, formamide must be replaced by water if the RNA is to be used in any in vitro enzymatic reaction.
To avoid RNA degradation as a result of multiple freeze thawing, it is recommended to store the RNA in small aliquots rather than in one single volume.
As an alternative to storing in solution, RNA can also be kept frozen or even at room temperature as a precipitate that is often obtained anyway at the end of the extraction procedure. Aliquots are removed from the precipitate after vortexing, and the RNA is pelleted by centrifugation and redissolved under RNase-free conditions immediately before use.
Storage of Samples Before RNA Extraction Under RNA Stabilizing Conditions
Samples containing the RNA of interest can also be stored prior to RNA extraction. Tissue or cells are allowed to be perfused by chemicals that inhibit RNase activity by denaturing all proteins. A rapid inactivation of enzymatic activity in the sample is required, in particular, in gene expression studies where any induction or repression of mRNA transcription because of changed environmental conditions must be prevented immediately after specimen collection to reflect the expression profile of the intact tissue.
It has repeatedly been shown that Trizol®, a mono-phasic solution of phenol and guanidinium isothiocyanate (Invitrogen Life Technologies, Carlsbad, CA), can be used to store samples for extended periods of time even at ambient temperatures, without a significant decrease of the quantity or quality of RNA.[6,7] Therefore, storage of samples in Trizol should be considered whenever samples cannot be kept frozen immediately after collection.
Recently, another commercially available product, RNAlater® (Ambion, Austin, TX), has been introduced on the market. RNAlater® is a patented aqueous, nontoxic tissue and cell storage reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue and cell samples by quickly permeating the tissue. It allows to store collected tissue specimens and cells at 4°C or even at ambient temperature, eliminating the need for 1) immediate snap-freezing of tissue in liquid nitrogen after collection and 2) instant RNA extraction. Most of the commonly used techniques can be used to extract RNA from RNAlater®-treated samples. RNAlater® has been shown to be suitable for RNA stabilization in animal and plant tissue and cells as well as in bacteria. Further information is available from Ambion's Web site at http:// www.ambion.com.
Tissue, cell, and serum samples can also be stored in a native, unprocessed form. However, to block RNA degradation as soon and as efficiently as possible, specimens should be frozen immediately after collection, and thawing and refreezing before RNA extraction must be avoided. It has been shown repeatedly that handling and storing conditions of diagnostic samples have a significant impact on the RNA levels detected by RT-PCR. Tissue samples collected for gene expression studies should be snap-frozen in liquid nitrogen. All samples should be stored at — 70C at all times.
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