Ten Sources of RNase Contamination
Living with RNase
Bodily fluids such as perspiration are rich in RNase activity (we jokingly refer to RNases from these sources as "fingerases"). Thus, the use of ungloved hands could easily result in RNase contamination compromising critical experiments. Use gloves and make it a point to discard used gloves and don a new pair frequently during experiments.
Tips & Tubes
Tips and tubes can be an easily overlooked source of RNase contamination. Merely autoclaving will not destroy all RNase activity, since these enzymes are very robust and can regain partial activity upon cooling to room temperature. Always use tips and tubes that have been tested and certified RNase-free. Ambion has a broad selection of certified RNase-free tips and tubes.
Water and Buffers
Due to the ubiquitous nature of RNases, the water and buffers used in molecular biology applications can be frequent sources of RNase contamination. DEPC-treatment is the most common method used to inactivate RNases in water and buffers. However, certain reagents such as Tris cannot be DEPC-treated. Ambion provides a variety of buffers, and water (DEPC and non DEPC-treated), that are guaranteed RNase-free. An alternative to DEPC-treatment, RNAsecure, is also available that can be used to treat primary amine solutions such as TRIS and does not require a 2 hr treatment nor autoclaving.
Contact with laboratory benchtops, glassware, plasticware, and other surfaces that are left exposed to the environment can lead to the introduction of RNase contamination into crucial experiments. These surfaces get contaminated due to the presence of bacterial and fungal spores present in many laboratory environments. Likewise, dead cells shed from human skin can also lead to contamination of exposed surfaces. These surfaces can be treated with an RNase decontamination solution such as Ambion's RNaseZap. Glassware and metalware can be baked overnight (6 to 8 hours is sufficient) at 450°F to inactivate RNases.
All tissue samples contain endogenous RNases. In lieu of immediate processing, liquid nitrogen is often used to rapidly freeze tissues after harvest to minimize RNA degradation. However, freezing tissue in liquid nitrogen is not always convenient, especially if large numbers of samples need to be preserved. Ambion's RNAlater™ is a tissue storage and stabilization solution that preserves RNA within tissues and cells. The pieces of tissue can be simply dropped into 5 to 10 volumes of RNAlater and stored at 4°C for up to a month prior to RNA isolation.
Small amounts of RNases that may co-purify with isolated RNA can compromise downstream applications. Such contamination can also be introduced from tips, tubes, and other reagents used in these procedures. RNase inhibitors are commonly used as a precautionary measure in most enzymatic manipulations of RNA to keep such contaminants in check. Ambion's SUPERase•In™ is the only RNase inhibitor that inhibits RNases A, T1, and RNase 1 (placental ribonuclease inhibitor (RI) only inhibits RNase A).
Plasmid DNA used for in vitro transcription and coupled transcription:translation reactions can introduce RNase contamination into reactions. Many researchers degrade the RNA in plasmid preps by RNase treatment. If this procedure has been used, Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. If the DNA template has been linearized by restriction enzyme digestion, a similar treatment is recommended, since restriction enzymes may be contaminated with RNases.
The presence of trace amounts of RNase can compromise RNA integrity even if the samples are stored frozen in an aqueous environment. The best method to preserve isolated RNA for long-term storage is to perform a salt/alcohol precipitation and store the nucleic acid as a precipitate in this solution. The low temperature and the presence of alcohol inhibit all enzymatic activity. The lower than neutral pH (due to the presence of sodium acetate or ammonium acetate) also helps stabilize the RNA. Note that the RNA will have to be centrifuged out of this solution prior to any downstream application. Another option is to resuspend the RNA in Ambion's RNAsecure™ Resuspension Solution, which contains an RNase-inactivating reagent. After the addition of RNAsecure, simply heat the sample at 60°C for 10 min to inactivate any RNases. If contamination of the sample is suspected at a later date, re-heating will inactivate any new contaminants.
Though RNase contamination is most commonly suspected whenever RNA degradation is observed, RNA molecules can also undergo strand scission when heated in the presence of divalent cations such as Mg2+ or Ca2+ at >80°C for 5 minutes or more. Thus, a chelating agent should be present whenever there is a requirement for heating RNA. THE RNA Storage Solution has been expressly designed for storing RNA. It contains 1 mM sodium citrate, which is an efficient chelator of divalent cations, and has a relatively low pH (~6.4) that minimizes RNA base hydrolysis.
Both commercially purchased and laboratory prepared enzymes can be a potential source of RNase contamination. At Ambion, we have used RNaseAlert (Cat. #1964) to determine the extent of RNase contamination in numerous commercially available enzymes (For more information, read "Is Your DNase RNase-Free?". It is important that you use only enzymes that are RNase-free when working with RNA. Ambion provides RNase-free enzymes for different kinds of RNA analysis experiments.