Precautions for Handling of RNA
Nature of RNA
In contrast to DNA, RNA is a single-stranded polynucleotide that is very susceptible to degradation by base- or enzyme-catalyzed hydrolysis. Working with RNA is more demanding due to both the chemical instability of RNA and because of the ubiquitous presence of RNases. Further, unlike DNases which require metal ions for activity, RNases have no requirement for metal ion co-factors and can maintain activity even after prolonged boiling or autoclaving.
RNases are found everywhere, including on laboratory workers´ hands and in airborne microorganisms. Special precautions must be taken when working with RNA. All reagents and equipment must be specially treated to inactivate RNases prior to use. Below are some tips to help ensure that your laboratory environment is as clean as possible.
Tips for Maintaining an RNase-free Environment
Gloves and contact
When working with RNA, wear gloves at all times. After putting on gloves, avoid touching contaminated surfaces and equipment with the gloved hands. Even if all the reagents have been decontaminated, RNases can be reintroduced by contact with ungloved hands or with unfiltered air.
Equipment and disposable items
Use sterile, disposable plasticware whenever possible. These require no treatment and are considered to be RNase-free. Electrophoresis tanks for RNA analysis can be cleaned by wiping them with a solution of SDS (1%), rinsing with water, then rinsing with absolute ethanol, and finally soaking them in 3% H2O2 for 10 minutes. Rinse tanks with DEPC (diethyl pyrocarbonate)-treated and autoclaved water before use (see below).
Glassware and plasticware
Treat glassware and plasticware with RNase-inactivating agents. Glassware should be baked at +180°C for at least 4 hours. Note, however, that autoclaving alone is not sufficient to eliminate RNases from your experiments. Soak plasticware (2 hours, +37°C) in 0.1 M NaOH/1 mM EDTA (or absolute ethanol with 1% SDS), rinsed with DEPC or DMPC (dimethyl pyrocarbonate) treated water and heated to +100°C for 15 minutes in an autoclave.
To treat water with DEPC (or DMPC, dimethyl pyrocarbonate, a less toxic alternative to DEPC that can be used in the same manner as DEPC), first incubate it with DEPC (2 hours, +37°C) and then autoclave it to hydrolyze any unreacted DEPC.
Workspace and working surfaces
Designate a special area for RNA work only. Treat surfaces of benches and glassware with commercially available RNase inactivating agents. Also, wipe benches with 100% ethanol each time prior to use, in order to rid the area of microorganisms.
Whenever possible, purchase reagents that are free of RNases. Be sure to separate reagents used for RNA work from "general use reagents" in the laboratory. All solutions, except Tris buffers, should be treated with 0.1% DEPC (or DMPC) overnight at room temperature and then autoclaved. Autoclaving hydrolyzes and destroys unreacted DEPC and DMPC. Alternatively, solutions can be made with DEPC-treated and autoclaved water in RNase-free glassware. Tris reacts chemically with DEPC and DMPC, and therefore, solutions of Tris cannot be made RNase-free using DEPC or DMPC. Dedicate one bottle of Tris for RNA work only. Use baked spatulas and glassware and DEPC/DMPC-treated water for making the buffers.
Note: Autoclaving without DEPC/DMPC treatment is insufficient for inactivating RNases.
Handling of fresh and stored material before extraction of RNA
Extract RNA as quickly as possible after obtaining samples. For the best results, use either fresh samples or samples that have been quickly frozen in liquid nitrogen and stored at -70°C. RNA in inadequately maintained samples can be degraded by intracellular nucleases, specifically in tissues that are rich in nucleases (such as spleen and pancreas).
RNase inhibitors can be used to protect RNA from degradation during both isolation and purification and also in downstream applications such as reverse transcription into cDNA by RT-PCR, in vitro RNA transcription/translation reactions and RNA-dependent in vitro functional assays.
Protector RNase Inhibitor
Protector RNase Inhibitor is one of the best characterized RNase inhibitors, effective on a wide spectrum of RNases (RNase A, RNase B, RNase T2) and offering the best protection of precious RNA samples. Protector RNase Inhibitor is fully active over a broad temperature range of +25 to +55°C. Even at +60°C some RNase inhibition is still measured. This is advantageous when performing reverse transcription reactions at elevated temperatures to overcome secondary structure in RNA, or when working with thermostable reverse transcriptases like Transcriptor Reverse Transcriptase. To keep the inhibitor active, avoid temperatures above +60°C, solutions containing strong denaturing agents (such as SDS or urea), and maintain reducing conditions (1 mM DTT). To protect difficult RNA samples, the amount of Protector RNase Inhibitor can be increased up to 16 times the standard concentration without interfering with the performance of enzymes used in the assay.
Other inhibitors include Macaloid and Vanadyl-ribonucleoside complexes.
Storage of RNA
Store RNA at -70 to -80°C, as aliquots in ethanol or isopropanol. Most RNA is relatively stable at this temperature. Centrifuge the RNA and resuspend in an appropriate RNase-free buffer before use.
Drying, dissolving, and pipeting RNA
RNA can be dried briefly at +37°C or in a vacuum oven. When working with RNA, place all samples on ice. For the reasons mentioned above, RNA is very susceptible to degradation when left at room temperature. Dissolve RNA by adding RNase-free buffer or water, then standing the tube on ice for 15 minutes. Gently tap the tube or use vortexing with caution.
Although DNA is relatively stable at elevated temperatures (+100°C), most RNA is not (except for short RNA probes, which are stable for 10 minutes at +100°C). Therefore, avoid high temperatures (above +65°C) since these affect the integrity of the RNA. Instead, to melt out secondary structures, heat RNA to +65°C for 15 minutes in the presence of denaturing buffers.