RNA Technical Tips
Hand and Fingers:
Bodily fluids such as perspiration are rich in RNase activity. Therefore, the use of ungloved hands often result in RNase contamination compromising critical experiments. Use gloves and make sure to discard them and don a new pair frequently during experiments. Do not touch any surface with ungloved hands that will come into contact with your sample.
Tips and Tubes:
Tips and tubes can be a source of RNase contamination. Merely autoclaving will not destroy all RNase activity, since these enzymes are very stable and can regain partial activity upon cooling to room temperature. Always use tips and tubes that have been tested and certified RNase-free. BioWorld has a broad selection of certified RNase-free tips and tubes.
Water and Buffers:
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 from water and buffers. However, certain reagents such as Tris cannot be treated with DEPC
Contact with laboratory bench tops, glassware, plastic ware, and other surfaces that are left exposed to the environment can lead to 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 and body fluid contact. 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 BioPlus RNase Out (tm) (catalog # 640200). Glassware and metal ware can be backed at 450F to inactivate RNase.
All organisms contain RNases. 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. BioWorld offers a tissue storage stabilization solution that preserves RNA within tissues and cells. The pieces of tissue can be simply dropped into 5-10 volumes of RNASafe (tm) and stored at 4C for the isolation of RNA at a later time.
Trace amounts of RNases that may co-purify with isolated RNA can degrade the RNA that interests you. RNase inhibitors are commonly used as a precautionary measure in most enzymatic manipulations of RNA.
Plasmid DNA used for in vitro transcription or 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 we recommend a Proteinase K treatments followed by a phenol: chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. If the DNA template has been cut by restriction enzyme digestion, a similar treatment is recommended, since restriction enzymes may be contaminated with RNases.
Commercially purchased enzymes can be a potential source of RNase contamination. We use RNase/Alarm(tm) to determine the extent of RNase contamination in numerous commercially available enzymes.
The best method to preserve isolated RNA for a long-term storage is to perform a salt/alcohol precipitation and store and 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 pH 5.2) also helps to stabilize the RNA. Another option is to resuspend the RNA in a BioPlus RNA SAFE, which contains RNase inactivating reagent. After the addition of RNA Safe, heat the sample at 60C for 10min to inactivate RNases
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 a divalent cation such as Mg or Ca at > 80C for 5 minutes or more. Thus a chelating agent should be present whenever there is a requirement for heating RNA. RNA Safe has been designed for storing RNA. It contains 1mM sodium citrate, pH 6.4, which is an efficient chelator of divalent cations, and minimizes RNA base hydrolysis.