To the average molecular biologist working with RNA, preventing, detecting and eliminating nuclease contamination, particularly ribonuclease (RNase) contamination, is a constant and somewhat annoying challenge. The following discussion pinpoints some of the sources of RNase contamination and highlights ways to eliminate contamination problems at various stages while working with RNA.

RNases Are Ubiquitous and Sturdy Enzymes

Because they play an important role in nucleic acid metabolism, RNases are found in both prokaryotes and eukaryotes, and in practically every cell type. The human body uses RNases to defend against invading microorganisms by secreting these enzymes in fluids such as tears, saliva, mucus and perspiration. The primary source of RNases within most environments, however, is microorganisms — namely bacteria, fungi and their spores. In a laboratory setting, RNase contamination problems are exacerbated since microorganisms and their derivatives (e.g., restriction enzymes, polymerases, etc.) are frequently used as essential tools or may even be the object of scientific study. As you might imagine, these proteins can be found practically everywhere in a molecular biology laboratory!

RNases, especially those belonging to the RNase A family, are fairly small, compact proteins containing several cysteine residues that form numerous intramolecular disulfide bonds. As a result, denatured RNases tend to regain their native structure and partial function after being cooled to room temperature in the absence of a denaturant. Consequently, RNases can retain activity after freeze-thaw cycles and even autoclaving (see Technical Bulletin 178). The robust nature of these enzymes makes them refractory to many methods of decontamination. Indeed, drastic chemical methods are often required to eliminate RNases from surfaces and solutions.

Preventing RNase Contamination

Basic Precautions
Some basic precautions need to be taken when working with RNA. These include:

1. wearing gloves throughout experiments to prevent contamination from RNases found on most human hands (we jokingly refer to these as "fingerases"),
   
   
2. changing gloves after touching skin (e.g., your face), door knobs, and common surfaces,
   
   
3. having a dedicated set of pipettors that are used solely for RNA work,
   
   
4. using tips and tubes that are tested and guaranteed to be RNase-free,
   
   
5. using RNase-free chemicals and reagents, and
   
   
6. designating a "low-traffic" area of the lab that is away or shielded from air vents or open windows as an "RNase-free Zone".

These common sense precautions will go a long way towards minimizing RNase contamination problems.

Surfaces
Laboratory surfaces, such as benchtops, centrifuges and electrophoresis equipment, should be assumed to be contaminated with RNases, since they are usually exposed to the environment. Eliminating contamination from these surfaces is fairly easy and can be done rapidly. Prior to using any shared laboratory equipment or benchtops, make it a point to decontaminate the surface by wiping it down with a commercially available RNase decontamination solution such as Ambion's RNaseZap™ (see below), or a mild solution of bleach or NaOH. Using a detergent solution like the kind used for soaking and cleaning glassware will not be as effective as these solutions. Detergent solutions may actually exacerbate the problem, since detergents can spread the RNase contamination over a wider area.

RNaseZap is a combination of three different chemicals that will completely inactivate RNases (and any other enzyme) immediately upon contact. Simply spray the surface with the solution and then rinse it thoroughly with nuclease-free water. RNaseZap Wipes, which are towelettes pre-soaked with the RNaseZap, are particularly convenient for decontaminating pipettors, benchtops and other surfaces. ElectroZap™, a specially optimized formulation for pH meters, can be used to eliminate contamination from the surface of pH electrodes.

Glassware and Metalware
Glassware and metalware can be treated with RNaseZap. An alternative is to bake these items, which may be more convenient if large numbers of items are involved. The baking procedure typically involves incubation in an oven at 450°F for 2 hours or more. (Don't include tape as it will burn!) Prior to baking, be sure to wrap the metalware items and the tops of beakers and flasks with aluminum foil to prevent contamination after baking. When 2 hours is too long to wait, treating with RNaseZap is a great alternative. Just make sure that the RNaseZap reagent is not in contact with surfaces of forceps, spatulas or other reactive metalware (e.g., aluminum) for more than a few minutes, as this could lead to corrosion. Mark baked and RNaseZap-treated items as "RNase-free" to distinguish them from untreated pieces. We recommend storing the treated equipment in a clearly marked "RNase-free Zone" to prevent accidental contamination.

Tips & Tubes
Tips and tubes are an easily overlooked source of RNase contamination. We typically do not find it necessary to decontaminate microfuge tubes used for RNA work if they are from unopened or carefully handled bags. However, we do consistently find a small percentage of tubes from other vendors (even those marketed as RNase-free), that cause RNA degradation. As explained earlier, simply autoclaving tips and tubes does not ensure elimination of RNases because these enzymes are very robust and will regain partial activity after cooling to room temperature (see Technical Bulletin 178).

When working with solutions or enzymes that might be contaminated with RNases, use RNase-free barrier (filter) pipet tips to prevent cross-contamination of your RNA samples or RNase-free solutions. Ambion provides RNase-free tips (including filter tips) of many sizes that are compatible with most commonly used pipettors. We also supply regular and non-stick microfuge tubes, which can be used for any molecular biology experiment. Each lot of tips and tubes is rigorously tested for RNase and DNase contamination and is certified nuclease-free.

Using RNase-free Solutions
Buffers and solutions are a common source of RNase contamination. Using RNase-free buffers and solutions is always the best approach when working with RNA. As discussed earlier, merely autoclaving prepared solutions is not sufficient for eliminating RNase contamination. Solutions need to be treated with DEPC or with an RNase inactivation reagent such as RNAsecureª (see the next section, "Preparing RNase-free Solutions", for more information).

Ambion provides a variety of prepared RNase-free buffers and reagents for use with RNA. All buffers are subjected to rigorous quality control procedures and are guaranteed RNase- and DNase-free.

Preparing RNase-free Solutions

DEPC Treatment
Diethylpyrocarbonate (DEPC) treatment is the most commonly used method for eliminating RNase contamination from water, buffers, and other solutions. (Note: DEPC cannot be used with certain buffers. See the below paragraph and "Alternatives to DEPC".) DEPC destroys enzymatic activity by modifying -NH, -SH and -OH groups in RNases and other proteins. The treatment typically involves incubating the solution at room temperature with 0.1% DEPC for a few hours, usually overnight, followed by autoclaving the solution to eliminate residual DEPC. A common concern that researchers have is the sweet, "fruity" aroma detected after autoclaving DEPC-treated solutions. When DEPC breaks down during autoclaving, a small amount of ethanol is produced. The ethanol can combine with trace amounts of carboxylic acid to produce volatile esters, which give off this characteristic smell. This is not a sign of incomplete DEPC removal and it will not interfere with any subsequent reactions.

Reagents containing primary amine groups (e.g., Tris) and some reagents containing secondary or tertiary amines (e.g., HEPES) cannot be DEPC-treated. The amine groups tend to react with and "sop up" the DEPC, making it unavailable for inactivating RNases (see Technical Bulletin 178). Also, modification of the reagent's amine groups could affect its buffering capability. Solutions that cannot withstand autoclaving and thus need to be filtered, such as MOPS, also cannot be DEPC-treated since autoclaving is essential for inactivating DEPC.

Alternatives to DEPC
Ambion's RNAsecure™ Reagent can be used as a convenient, noncarcinogenic alternative to DEPC for treating small volumes of precious reagents and solutions such as Tris and MOPS that cannot be DEPC treated. The reagent is supplied as a 25X stock. After the RNAsecure stock is diluted into the solution, the solution is heated to 60°C for 10 minutes, which "activates" the reagent. Unlike DEPC, which does not inactivate RNases introduced post-treatment, RNAsecure-treated solutions can be reheated to eliminate new contaminants. For resuspension of RNA pellets, Ambion also offers the RNAsecure Resuspension Solution, which is supplied at a 1X working concentration.

Detecting RNase Contamination

Buffers, Solutions and Surfaces
Tracing and identifying the source of RNase contamination can be a frustrating and time-consuming endeavor. The easiest but most expensive solution to the problem, especially if a reagent is suspected as the source of the contamination, is to discard all the existing stocks of reagents and start with fresh, RNase-free batches.

Another option is to test the suspected solutions for the presence of RNases. Testing can be performed by incubating a radiolabeled (³²P) RNA probe with the reagent(s) in question, analyzing the probe on a denaturing acrylamide gel, and looking for signs of probe degradation. Using this method, one should be able to detect as little as 0.1 pg/ml of RNase A (see Technical Bulletin 166). The same technique can also be easily adapted for detecting RNase contamination in tips, tubes, glassware or any other surface. However, this method can be quite laborious, expensive and demanding given the variety of buffers and reagents used for each molecular biology experiment and the number of surfaces involved.

Ambion provides a rapid and convenient alternative for RNase detection in the RNaseAlert™ Kits. These kits use a fluorescence-based assay involving the cleavage of an RNA oligonucleotide to which a fluorophore and a quencher are attached. In the presence of RNase, the oligonucleotide is cleaved and the fluorophore is separated from the quencher, resulting in a fluorescence signal. As little as 0.5 pg (4 x 10^-7 Units) of RNase A can be detected in 10 to 30 minutes using this kit. The RNaseAlert Kit is available both in single-tube and 96-well formats.

RNA Samples
RNases can be introduced into RNA samples during RNA isolation (e.g., when small amounts of RNases are carried over into the preparation) or during normal day-to-day use, which inevitably leads to repeated opening/closing of sample tubes and insertion of possibly contaminated pipet tips. RNase contamination is generally detected by looking for degradation of the RNA within the sample.

Total RNA. Total RNA samples can be analyzed by running the sample (2 to 5 µg) on a denaturing agarose gel and staining with ethidium bromide (Figure 1). Samples (as little as 1 µl of a 10 ng/µl RNA) can also be analyzed on the Agilent 2100 bioanalyzer (Agilent Technologies) using an RNA 6000 LabChipå (Caliper Technologies Corporation; Figure 2). Intact total RNA samples should exhibit a 2:1 ratio of 28S to 18S ribosomal RNA band intensities. A ratio significantly lower than 2:1 is usually a sign of degradation.

Figure 1. Intact vs. Degraded RNA. Two µg of degraded total RNA and intact total RNA were run beside Ambion's RNA Millennium Markers™ on a 1.5% denaturing agarose gel. The 18S and 28S ribosomal RNA bands are clearly visible in the intact RNA sample. The degraded RNA appears as a lower molecular weight smear.

Figure 2. Agilent 2100 bioanalyzer Data. Electropherogram of a high quality, eukaryotic, total RNA sample. The 18S and 28S peaks are clearly visible at 39 and 46 seconds, respectively. The microchannels of the Bioanalyzer are filled with a sieving polymer and fluorescence dye. Samples are detected by their fluorescence and translated into electropherograms or into gel-like images (data not shown).

Poly(A) RNA. Assessing the integrity of poly(A) RNA samples can be accomplished by performing a Northern analysis using a probe against a housekeeping gene such as GAPDH, cyclophilin or ó-actin. Since the RNA sample is resolved by size on a denaturing gel, degradation manifests as a smear below the full-length message. The more severe the degradation, the lower molecular weight the smearing. Absence of a band/smear at the expected full-length message size or observation of a band/smear at the bottom of the gel is an indication that the RNA is very severely degraded.

Degraded RNA samples are unsuitable for Northern analysis, RACE protocols, or cDNA library construction. However, they might still be viable for RT-PCR, if small amplicons are being amplified, and for nuclease protection assays (NPAs), if the probe length is restricted to a small size.

Storing RNA

Samples Since RNA samples can be contaminated by small amounts of RNases carried over during RNA isolation or due to repeated sample use, proper storage can help minimize these problems.

Short-term Storage
For short-term storage, RNA samples can be resuspended in water or buffer and stored at -80°C. If water is the preferred medium, be sure to use nuclease-free water. Using a buffer solution that contains a chelating agent is a better way to store RNA. Chelation of divalent cations such as Mg⁺² and Ca⁺² will prevent heat-induced strand scission (RNA can be chemically cleaved when heated in the presence of Mg⁺²). Ambion provides nuclease-free water and a variety of buffers, including TE, 0.1 mM EDTA and THE RNA Storage Solution (which has the added benefit of a low pH), for storing RNA. All are subjected to rigorous quality control procedures and guaranteed to be nuclease free.

Long-term Storage
For long-term storage (more than a few weeks), RNA samples are best stored as a salt/ethanol slurry. To do this, take the RNA through all the steps of a regular precipitation with salt (e.g., 1/10 volume of 3 M NaOAc, pH 4.8) and ethanol (2 volumes of 100% ethanol) and store the mixture at -80°C without pelleting the RNA out of solution. The combination of low pH, low temperature and high alcohol content will stabilize the RNA and inhibit all enzymatic activity. Other alternatives for long-term storage are to store the RNA in formamide (Chomczynski, 1992) or to store the RNA in frozen aliquots at -20°C or below. If stored in formamide or ethanol, the RNA will need to be pelleted out of solution prior to quantitation or other manipulation.

Inhibiting RNases in Enzymatic Reactions

The traditional method for combating RNases in enzymatic reactions such as in vitro transcription, reverse transcription and translation is to use human placental ribonuclease inhibitor (also known as RNase Inhibitor Protein, RI or hPRI). This protein is an inhibitor only of the RNase A family of ribonucleases, which includes RNases A, B, and C. The mode of inhibition is non-competitive, i.e., the protein does not destroy these RNases but binds them in a 1:1 ratio. A potential problem with the inhibitor is that it might be contaminated with the very RNases that it is designed to inhibit, due to co-purification of the nucleases. Extended incubation of contaminated preps of this protein can lead to slow release of the nuclease into enzymatic reactions (see "SUPERase•In™: The Right Choice for Protecting Your RNA"). Thus, even though this inhibitor can be useful in solving many ribonuclease contamination problems, it is not necessarily the best inhibitor available.

Ambion's SUPERase•In™ is a broad-spectrum RNase inhibitor that protects RNA against not only the RNase A family of nucleases but also against RNase T1 and I. It can be used as a better alternative to placental ribonuclease inhibitor (RI or hPRI) in any enzymatic reaction. Ambion also offers the RNAsecure™ Reagent, a non-enzymatic alternative for use in reactions such as in vitro transcription, RT-PCR and translation.

Reference

Chomczynski, P. (1992) Solubilization in formamide protects RNA from degradation. Nucleic Acids Res. 20: 3791-3792.