Working with RNA

Living with RNase

Most researchers are acutely aware of the risk of RNase contamination, and we do not want to belabor this point or cause undue worry. We do not routinely find it necessary to treat the microcentrifuge tubes used with RNA if they are from unopened bags or from bags in which care was taken to avoid contaminating the tubes. Yet we do consistently find a small percentage of tubes (even those marketed as being RNase-free), the use of which results in RNA degradation. We do recommend that gloves be worn when handling any reagents or reaction vessels. (Note: Gloves which have touched refrigerator handles, door knobs, or pipettors are not RNase-free.) When performing procedures that use RNases (eg. ribonuclease protection assays and plasmid purifications), care should be taken that pipettors are not contaminated by accident. One potential source of contamination is the metal tip ejector mechanism on the side of the pipettor. Removing the metal ejector bar when it is necessary to insert the pipettor into a larger vessel where the ejector could come into contact with the walls or contents of the vessel will eliminate this concern.

A. Detecting RNase
While contaminating RNase can result in a failed experiment, it is often difficult an time-consuming to determine which solution or piece of equipment is responsible. In Ambion's Quality Control Department, we use an extremely sensitive RNA probe stability assay to detect RNase contamination. This assay can be performed in your own lab to detect RNases and a protocol is provided in Technical Bulletin 166 to facilitate this. However this assay is time consuming and requires working with radioactivity. As an alternative, Ambion's RNaseAlert™ Kit (Cat #1964) allows researchers to identify contaminated reagents and equipment quickly, and nonisotopically. In the RNaseAlert Kit procedure, an optimized RNA oligonucleotide, double-labeled with both fluorescent and quenching moieties, is introduced as a target for any contaminating RNase. In the presence of RNase, the substrate is cleaved, releasing the fluor which then fluoresces. The fluorescence signal can be detected by eye or with a fluorometer.

B. Getting rid of RNase
If RNase contamination of reagents or equipment is suspected to be a problem, extra precautions may be necessary. Autoclaving tips, tubes and solutions is not sufficient to inactivate RNases. Glassware can be baked at 300°C for four hours and plasticware, tubes and most solutions can be DEPC-treated (see below). However, both procedures are time-consuming, and DEPC is both expensive and possibly carcinogenic. As an alternative, Ambion's RNaseZap™ (Catalog #9780) can be used to eliminate RNase from glassware, plastic surfaces, countertops, and pipettors. RNaseZap™ has been shown to effectively inactivate 5 µg of RNase dried onto the bottom of eppendorf tubes without inhibiting subsequent enzymatic reactions performed in the same tube. The solution contains three ingredients known to be active against RNase. RNaseZap™ can be poured onto or wiped over surfaces and works immediately upon contact. Treated labware is simply rinsed twice with distilled water and is ready for use.

Treating Solutions with DEPC to Remove RNase

To ensure that solutions are free of RNase contamination, they can be treated with diethylpyrocarbonate (DEPC) [WARNING: DEPC is a suspected carcinogen: Take appropriate precautions when handling; e.g., always wear gloves and handle under an approved fume hood]. DEPC reacts with histidine residues of proteins and will inactivate RNases. However, it can also react with RNA, so it needs to be removed by heat treatment before the solution is used (DEPC breaks down to CO₂ and ethanol). Add DEPC to solutions at a concentration of 0.05 - 0.1% (e.g., add 0.5 - 1 ml DEPC per liter); stir or shake into solution, incubate for several hours; autoclave at least 45 minutes, or until DEPC scent is gone. Please be aware that compounds containing primary amine groups, such as Tris (2-Amino-2-hydroxymethyl-1,3-propanediol), will also react with DEPC, and thus should be added only after DEPC treatment is complete. Note: We have observed that distilled water, treated with DEPC and thoroughly autoclaved, caused a 20% inhibition of translation in a reticulocyte lysate. We find that distilled water is generally already RNase-free, and so does not need to be treated.

How to Store RNA

RNA may be stored in a number of ways. For short-term storage, RNase-free H₂O (with 0.1 mM EDTA) or TE buffer (10 mM Tris, 1mM EDTA) may be used. RNA is generally stable at -80° C for up to a year without degradation. Magnesium and other metals catalyze non-specific cleavages in RNA, and so should be chelated by the addition of EDTA if RNA is to be stored and retrieved intact. It is important to use an EDTA solution known to be RNase-free for this purpose (older EDTA solutions may have microbial growth which could contaminate the RNA sample with nucleases). It has been suggested that RNA solubilized in formamide may be stored at -20°C without degradation for at least one year (Chomczynski, 1992).

For long term storage, RNA samples may also be stored at -20°C as ethanol precipitates. Accessing these samples on a routine basis can be a nuisance, however, since the precipitates must be pelleted and dissolved in an aqueous buffer before pipetting, if accurate quantitation is important. An alternative is to pipet directly out of an ethanol precipitate that has been vortexed to create an even suspension. We have found, however, that while this method is suitable for qualitative work, it is too imprecise for use in quantitative experiments. RNA does not disperse uniformly in ethanol, probably because it forms aggregates; non-uniform suspension, in turn, leads to inconsistency in the amount of RNA removed when equal volumes are pipetted.

How to Precipitate RNA

A. Precipitating with alcohol
Precipitating RNA with alcohol (ethanol or isopropanol) requires a minimum concentration of monovalent cations (for example: 0.2 M Na+, K+; 0.5 M NH₄+) (Wallace, 1987). After the salt concentration has been adjusted, the RNA may be precipitated by adding 2.5 volumes of ethanol or 1 volume of isopropanol and mixing thoroughly, followed by chilling for at least 15 minutes at -20° C. While isopropanol is somewhat less efficient at precipitating RNA, isopropanol in the presence of NH₄+ is better than ethanol at keeping free nucleotides in solution, and so separating them from precipitated RNA. RNA precipitation is faster and more complete at higher RNA concentrations. A general rule of thumb is that RNA concentrations of 10 µg/ml can usually be precipitated in several hours to overnight with no difficulty, but at lower concentrations a carrier nucleic acid or glycogen should be added to facilitate precipitation and maximize recovery.

B. Precipitating with lithium chloride
Lithium Chloride may also be used to precipitate RNA, and has the advantage of not precipitating carbohydrate, protein or DNA. LiCl is frequently used to remove inhibitors of translation which copurify with RNA prepared by other methods. A final LiCl concentration of 2-3 M is needed to precipitate RNA (adding an equal volume of 4 M LiCl, 20 mM Tris-HCl, pH 7.4, and 10 mM EDTA works well). Note that no alcohol is needed for LiCl precipitation. RNA should be allowed to precipitate at -20°C; precipitation time depends on RNA concentration. It is generally safe to allow the RNA to precipitate for several hours to overnight. After centrifugation to collect the RNA, pellets can be rinsed with 70% ethanol to remove traces of LiCl. LiCl efficiently precipitates RNA greater than 300 nt in length. While LiCl can effectively precipitate RNA from more dilute solutions, for best results, the RNA concentration should exceed 200 µg/ml.

Incorporation and Yield

"Percent incorporation" is calculated by comparing the amount of radioactivity incorporated into synthesized RNA with the total amount of radioactivity in the reaction. This is often done by TCA precipitation (see below) but can also be done by simply counting an aliquot of the transcription reaction before and after removal of unincorporated nucleotides. Note that the counts used for comparison must be adjusted to represent equivalent aliquots. Unincorporated nucleotides may be removed by precipitation using LiCl or NH₄OAc and EtOH (see above), by passing the transcription reaction over an RNase-free Sephadex column (e.g., Ambion's NucAway™ column), or by gel purification.

The amount of radioactivity incorporated into RNA may also be determined by precipitation with trichloroacetic acid (TCA), filtration, and counting in a liquid scintillation counter. Add a 2 µl aliquot of an RNA labeling reaction to 98 µl of water containing 10 µg of carrier DNA or RNA. To this add 2 ml of cold 10% TCA, vortex and incubate on ice 5 minutes. Collect the precipitate by filtering under vacuum through GF/C glass fiber filters. Wash the sample tube twice with 2 ml 10% TCA and once with 2 ml of 95% ethanol, passing the washes through the filter. After drying, these filters may be placed in vials with liquid scintillation cocktail and counted. Note: Both RNA and DNA may be precipitated using this method.

Since percent incorporation of a radiolabeled nucleotide is directly proportional to yield, the actual yield of a transcription reaction is equivalent to that proportion of the theoretical maximal yield. For example, Ambion's MAXIscript™ Kit reactions have a theoretical 100% yield of 77 ng when the transcription reaction contains a limiting nucleotide concentration of 3 uM. Therefore, if for a given reaction the percent incorporation was 80%, then 0.80 X 77 ng or 62 ng of labeled RNA were synthesized.

Some ribosomal subunit size relationships within the eukaryotes are illustrated in Table 1. Both 18S and 28S rRNA contain modified nucleotides, including methylated ribose and pseudouridine (46 and 37 for 18S; 71 and 60 for 28S, respectively) .

RNA Size Markers

Ambion offers several different ranges of RNA size markers that can be obtained unlabeled for staining with EtBr or biotinylated for subsequent secondary detection. The RNA Century Marker Set (Cat #7140 - unlabeled, #7175 - biotinylated) contains 5 transcripts evenly spaced between 100 -500 nt, which are ideal for ribonuclease protection assays and gel purification of RNA probes. The RNA Century Markers can also be obtained as DNA templates (Cat #7780 and 7782) for the synthesis of radiolabled RNA markers in an in vitro transcription reaction. Ambion's RNA Millennium Marker Set (Cat #7150 - unlabeled, #7170 - biotinylated) contains 10 transcripts ranging from 0.5-9.0 kb for use with Northern analysis.

RNA transcripts and double-stranded DNA markers (e.g. pUC 19/Hpa II, Cat #7760 and #7770) can also be end-labeled with polynucleotide kinase (5' end-labeling reaction) or Klenow Fragment (3' filling reaction) and denatured, for use as labeled size markers.

Other guides to RNA size and migration position are the xylene cyanol and bromophenol blue dyes present in most loading buffers, and rRNA species present during electrophoresis of total RNA for Northern analysis. The migration position of the dyes included in loading buffers is affected both by gel percentage and composition (denaturing vs. nondenaturing). Ribosomal RNA comprises 80% of total RNA samples. Both the 18S and 28S species are strongly visible in Northern gels stained with EtBr or UV-shadowed. The table above gives their sizes in several different vertebrate species.

References

• Chomczynski, P. (1992) Solubilization in formamide protects RNA from degradation. Nuc. Acids Res. 20:3791-3792.
• Wallace, D.M. (1987) Precipitation of Nucleic Acids. Methods of Enzymology 152:41-46.

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